Koch Lab:Data/Steve grad data set notes (partial)

010326.txt
D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0025.dat	Contains example of 0.35 um peeler. Otherwise mostly junk

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0026.dat	Contains example of 0.53 um in BsaIBuffer

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0027.dat	Pre-BsaI, shows some examples of artifactual data

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0028.dat	Some Pre-BsaI, no good data

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0029.dat	Trying to gather Pre-BsaI data. Large File.

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0030.dat	BsaI--contains one good template

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0031.dat	BsaI--really contains no good templates

010327.txt
010327



Step 0 :  Peel Buffer + Tween ;  5.000000  s.v.;   0.000000  min.

Step 1 :  Peel Buffer ;  5.000000  s.v.;   0.000000  min.

Step 2 :  antidig 20 ug / ml in PeelBuf ;  1.000000  s.v.;   11.000000  min.

Step 3 :  BGB (old aliquots) ;  1.000000  s.v.;   5.000000  min.

Step 4 :  BGB (repeat) ;  1.000000  s.v.;   5.000000  min.

Step 5 :  PeelBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  17-mer (most recent batch, 1:10 in PBS, 1:3 in PeelBuf) ;  1.000000  s.v.;   10.000000  min.

Step 7 :  EcoRI Bufferf* ;  5.000000  s.v.;   0.000000  min.

Step 8 :  Beads, Bangs 0.53 micron streptavidin, 1:10 into EcoRI Buffer* ;  1.000000  s.v.;   20.000000  min.

Step 9 :  EcoRI Buffer* ;  10.000000  s.v.;   0.000000  min.

Step 10 :  *1 sample with tween, the other without ;  0.000000  s.v.;   0.000000  min.



Non-tween sample has lots of stuck beads (about 20 or more per octagon, and less than 1 tether)

Tween sample had less stuck beads. Had more things that looked like tethers, but nothing peeled! FUUCK!

Also, there is some kind of problem with the oscilloscope DAQ board, and channel-to-channel interference...probably a grounding issue

All of the data are fucking worthless

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0032.dat	non-tween sample. both segments are garbage. sample sucks

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0033.dat	tween sample...stopped early to change conditions

010328.txt
010328



Step 0 :  Dopes ;  5.000000  s.v.;   0.000000  min.

Step 1 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 2 :  antidig 20 ug / ml in PBS ;  1.000000  s.v.;   10.000000  min.

Step 3 :  BGB (old aliquots) ;  1.000000  s.v.;   5.000000  min.

Step 4 :  BGB (repeat) ;  1.000000  s.v.;   15.000000  min.

Step 5 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 6 :  17-mer (1:10 aliquot) ;  1.000000  s.v.;   10.000000  min.

Step 7 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 8 :  Bangs 0.53 Streptavidin 1:10 in Dopes ;  1.000000  s.v.;   20.000000  min.

Step 9 :  Dopes ;  10.000000  s.v.;   0.000000  min.

Step 10 :  Delay..maybe 1/2 hour ;  0.000000  s.v.;   0.000000  min.

Step 11 :  1%BsaI in 90% dopes + 10 mM EDTA ;  5.000000  s.v.;   0.000000  min.



After 10 minutes of bead incubation, before washing beads away, there are about 1 or two possible tethers per 90 micron octagon. Since I am using 3x more 17-mer than I was yesterday, this could partially explain my difficulty.

Also, I notice that the bangs beads are very clumpy. This could be a factor in reducing the tether density.

After washing away beads, seems to be about 3 beads or whatever. But I notice that they transiently stick--this is also a problem.

The sample glass is actually fairly dirty.

Added BsaI in 90%Dopes + 10 mM EDTA at 11:42PM

VIDEO of little dirt farm 12:33 AM

SO, Here is my guess as to what the secrets were to tonight:

A. Use higher DNA concentration...makes a big deal compared to background stuck beads?

B. Revert to old buffers. Same consideration as A, perhaps sticking more of a problem with the other buffers.

C. Do I have DNAse or other contamination??? It appeared to me that the tethers were going away with time, and becoming more prone to breaking. After loading in the BsaI and EDTA buffer, the problem seemed to go away, and I got lots of good data.

Also, today, I fixed a bug related to offset subtraction.

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0034.dat		I think wiggle array messed up

In this file, segment 006, I took video on KochVII, which shows how a peeler looks as a tether. In addition, this video shows my z=8.5 focusing method. Right after that on video is example of transient sticking problem. Even though segment 6 and 7 break soon, I think the vidoe is a good example

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0035.dat		I think wiggle array messed up

Some great BsaI popping. I would say about 1/3 were good. Of those, for some reason they still seemed to break early

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0036.dat		Just one segment, which shows that even after all of this time, there are still BsaI poppers.

I HAVE FIXED THE OFFSET SUBTRACTION FOR THE FIND TETHER CENTER NOW>

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0037.dat		Has some really great extremely fast pulling (where the force may indeed be incrementally higher)

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0038.dat		Slow popper in segment 004. Forces were all over the board (high, low). Should stare at this one for a while

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0039.dat		One single fast popper?

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010327\0040.dat		at least four templates with extremely fast popping (50 MHz / sec). this seemed pretty awesome, but I guess you needed to be there.

010329.txt
3/29/01 2:55 AM



Step 0 :  Dopes ;  1.000000  s.v.;   10.000000  min.

Step 1 :  0.9xPBS 10 mM EDTA ;  5.000000  s.v.;   0.000000  min.

Step 2 :  antidig 20 ug / ml in 0.9xPBS + 10 mM EDTA ;  1.000000  s.v.;   11.000000  min.

Step 3 :  10 mg / ml BGB in 0.45x PBS 5 mM EDTA ;  2.000000  s.v.;   9.000000  min.

Step 4 :  10 mg / ml BGB in 0.45x PBS 5 mM EDTA ;  2.000000  s.v.;   5.000000  min.

Step 5 :  17-mer 1:11 in 10/11 PBS + 10 mM EDTA ;  1.000000  s.v.;   14.000000  min.

Step 6 :  Bangs 0.53 streptavidin 1:10 in final buffer 0.81 PBS 9 mM EDTA ;  1.000000  s.v.;   15.000000  min.

Step 7 :  0.9x Dopes + 10 mM EDTA ;  10.000000  s.v.;   0.000000  min.

Step 8 :  0.9x Dopes + 10 mM EDTA ;  10.000000  s.v.;   0.000000  min.

Step 9 :  0.9x Dopes + 10 mM EDTA ;  10.000000  s.v.;   0.000000  min.

Step 10 :  0.9x Dopes + 10 mM EDTA + 1:50 EcoRI at about 2:06 AM ;  5.000000  s.v.;   0.000000  min.



after first bead wash-away, there were 1.1 "look-like" tethers per 90 micron octagon. about the same or less stuck beads (that is, stuck beads are lower than before, most probably because of the higher casein concentration)

Given the about 30% success rate, I would say actual density is about 0.3 per octagon, which is definitely very low.

after second wash, SEEMS like fewer tethers. i counted 0.8 per octagon versus 1.6 stuck. however, peelers efficiency increases!

2:06 AM, Flowed in EcoRI in 0.9x Dopes + 10 mM EDTA (1 ul enzyme in 50 ul buffer, flowed in 50 ul)

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0041.dat	initial file. shows maybe 30% success rate

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0042.dat	demonstrates strange increase in peeling ability by washing a second time!

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0043.dat	0.5 MHz / sec

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0044.dat	5 MHz / sec

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0045.dat	10 MHz / sec

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0046.dat	20 MHz / sec

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0047.dat	50 MHz / sec

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0048.dat	free bead, maybe not configured correctly

BAD OFFSET SUBTRACTION

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0049.dat	free bead 50 MHz/ sec

BAD OFFSET SUBTRACTION

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0050.dat	50 MHz / sec no bead...messed up, though

BAD OFFSET SUBTRACTION

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0051.dat	0.5 MHz / sec no bead

BAD OFFSET SUBTRACTION

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0052.dat	50 MHz / sec no bead

BAD OFFSET SUBTRACTION

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0053.dat	50 MHz / sec free bead

BAD OFFSET SUBTRACTION

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0054.dat	50 MHz / sec fixed offset correction

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0055.dat	50 MHz / sec fixed offset correction

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0056.dat	0.5 MHz / sec EcoRI...lots of popping, but seeming no full-length

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0057.dat	5 MHz / sec EcoRI pops

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0058.dat	10 MHz / sec EcoRI pops

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0059.dat	20 MHz / sec

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010329\0060.dat	50 MHz / sec

010404.txt
4/4/01 3:31 AM



Step 0 :  Popping Buffer(04/03/2001) ;  5.000000  s.v.;   0.000000  min.

Step 1 :  Repeat ;  5.000000  s.v.;   0.000000  min.

Step 2 :  Antidig (Roche) 20 ug / ml diluted in Popping buffer ;  1.000000  s.v.;   10.000000  min.

Step 3 :  10 mg / ml BGB in 0.5 PBS + 5 mM EDTA ;  1.000000  s.v.;   10.000000  min.

Step 4 :  repeat ;  1.000000  s.v.;   10.000000  min.

Step 5 :  Popping Buffer ;  5.000000  s.v.;   0.000000  min.

Step 6 :  1:10 17-mer (+1mM EDTA today) in PBS ;  1.000000  s.v.;   10.000000  min.

Step 7 :  Popping Buffer ;  5.000000  s.v.;   0.000000  min.

Step 8 :  Bangs 0.53 1:10 into Popping Buffer ;  1.000000  s.v.;   20.000000  min.

Step 9 :  Popping Buffer (about 1AM) ;  10.000000  s.v.;   0.000000  min.

Step 10 :  delay ;  0.000000  s.v.;   83.000000  min.

Step 11 :  EcoRI, 1:100 into Popping Buffer (2:23 AM) ;  5.000000  s.v.;   0.000000  min.

Step 12 :  EcoRI, 1:100 (same as last batch) at about 3:04 AM ;  5.000000  s.v.;   0.000000  min.

Step 13 :  1:5 EcoRI into popping buffer 3:14 AM ;  2.000000  s.v.;   0.000000  min.

Step 14 :  Plain Popping Buffer (no enzyme) 3:22AM,  ;  5.000000  s.v.;   0.000000  min.



Had programming emergency for first hour. Figured out that it was a Splice12-segments(categorical) error. Resumed taking data.

15 tethers, selected by eye. 33% broke immediately, 4/15 peeled completely, and 9/15 peeled at least partially. So, I think 25% expectation for restriction enzymes is reasonable. Will now (2AM) add enzyme

Any secrets?

I think flow force may help lift good tethers off the surface. So that routine washing is a good thing.

For some reason I cannot get EcoRI to saturate

0062.dat	15 per-enzyme tethers for examples

0063.dat	0.5 MHz / sec

0064.dat	5 MHz / sec (lots of overstretching)

0065.dat	0.05 MHz / sec, several templates with popping

0066.dat	0.5 MHz / sec again, except this time surely at "room" temperature

0067.dat	0.5 MHz / sec again, with freshly added room temperature enzyme. LOTS of popping. THEORY: the flow force "rejuvinates" some stuck beads that are perfectly good tethers.

0068.dat	0.5 MHz / sec after adding 1:5 EcoRI

0069.dat	same as previous

0070.dat	0.5MHz/sec after washing with plain popping buffer

010405.txt
4/5/01 12:47 AM



Step 0 :  Popping Buffer, room temp ;  5.000000  s.v.;   0.000000  min.

Step 1 :  Antidig 20 ug / ml (1:10 in popping buffer) ;  2.000000  s.v.;   10.000000  min.

Step 2 :  BGB, same as yesterday ;  2.000000  s.v.;   10.000000  min.

Step 3 :  Pop Buffer ;  5.000000  s.v.;   0.000000  min.

Step 4 :  BGB ;  2.000000  s.v.;   10.000000  min.

Step 5 :  17-mer +EDTA (see yesterday) ;  1.000000  s.v.;   10.000000  min.

Step 6 :  Pop Buffer ;  10.000000  s.v.;   0.000000  min.

Step 7 :  Bangs 0.53 streptavidin 1:10 into Pop Buffer (Diluted yesterday...when flowing in, the color is non-homogenous, probably indicating clumping from the buffer?) ;  1.000000  s.v.;   10.000000  min.

Step 8 :  repeat ;  1.000000  s.v.;   10.000000  min.

Step 9 :  Pop Buff ;  10.000000  s.v.;   0.000000  min.

Step 10 :  BsaI 1:25 into Popping Buffer ;  1.000000  s.v.;   2.000000  min.

Step 11 :  Repeat above 5 times (ended about midnight) ;  0.000000  s.v.;   0.000000  min.

Step 12 :  seal nail polish ;  0.000000  s.v.;   0.000000  min.



tether density is very low (less than 1/2 per 90 micron)

I believe even more in the theory that good tethers transiently stick to the surface (I even grabbed one off and peeled it perfectly)

0071.dat	0.5 MHz / sec, lots of popping, and perhaps full poppers

0072.dat	same as previous. lots of segments shows the problem with biased force

0073.dat	5 MHz / sec, lots of popping

0074.dat	same as previous

0075.dat	10 MHz / sec Done for the night

010509.txt
5/9/01 2:23 AM



Step 0 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 1 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 2 :  antidig (Roche) 20 ug / ml in PBS ;  2.000000  s.v.;   7.000000  min.

Step 3 :  BGB 10 mg / ml in ? (same as last time) ; 2.000000  s.v.;   10.000000  min.

Step 4 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 5 :  DNA [either pRL574 peeling template from december, or 17 pM dsDNA PCR) ;  1.000000  s.v.;   14.000000  min.

Step 6 :  Dopes (ran out of thawed popBuf) ;  5.000000  s.v.;   0.000000  min.

Step 7 :  Bangs 1:10 in PopBuf 0.45 micron streptavidin, freshly diluted ;  1.000000  s.v.;   22.000000  min.

Step 8 :  PopBuf ;  10.000000  s.v.;   40.000000  min.

Step 9 :  EcoRI, 1:50 in PopBuf (LifeTech) (about 11:43PM) ;  2.000000  s.v.;   20.000000  min.

Step 10 :  EcoRI, 1:50 in PopBuf (LifeTech) (about 12:04PM) ;  2.000000  s.v.;   26.000000  min.

Step 11 :  EcoRI, 1:5 in PopBuf (LifeTech) (about 12:30PM) ;  0.000000  s.v.;   0.000000  min.



Data set 082, segment 005 through xxx, I was in some weird tether farm in the middle of the sample. It showed lots of wiggly beads (at least 10x higher conc. than other areas) and high probability of non-specific tethering, or double peeling tethering

12:30 AM added 1:5 Eco in PopBUf

also tonight, made 4 molal sucrose by adding .4 moles of sucrose to 100 ml of H2O (ends up about 200 ml). i then made 2 molal sucrose 50% popping buffer by mixing equal volumes.

0079

0080	EcoRI...peeling and some popping?

0081	EcoRI...peeling and some popping?

0082	0.5 MHz / s

freshly added Eco after 1st segment

0083	5 MHz / s

0084	0.5 Mhz / s

10x more EcoRI (1:5)

THIS IS THE BEST FILE YET...at least 3 popping events, which look to be at the correct site

0085	5 Mhz / s

EcoRI 1:5

This is an awesome data file!

NOTE: Mysterious fluctuations...possibly overstretch related...examine the shearing sequence for possible EcoRI binding...this gave me idea for using shearing to study protein binding!

0086	4.4 kb double tagged template in popBUf only. EVERYthing seems to break right at overstretch

0087	just for fun, added EcoRI 1:5...has a few overstretches

0088	overstretchin in 2 Molal sucrose in 50% popping buffer

0105092.txt
5/9/01 5:49 AM

3:10 added the sucrose

about 3:38 AM added 1:20 BsaI into 45% pop + 2 m Sucrose (45 ul popBuf + 50 ul 4 m Sucrose + 5 ul BsaI)

4:10 AM 1 m + 1:20 BSAI

4:29 AM 0.5 m + 1:20 BSAI

4:52 AM 0.25 m + 1:20 BsaI

5:12 AM 0.125 m + 1:20 BsaI

5:22 AM 0.0625 m + etc.

5:36AM 1:20 BsaI in popBuf (NO SUCROSE)

0089	0.5 MHz / sec

naked 17-mer in popBuf

0090	0.5 Mhz / sec

naked 17-mer in 2 m Sucrose

0091	0.05 Mhz / sec

naked 17-mer in 2 m Sucrose

0092	5 Mhz / sec

naked 17-mer in 2 m Sucrose

0093	0.5Mhz / sec

BsaI...lot's of overstretching!!!!

0094	0.05Mhz /sec

BsaI...still overstretching, but this time popping too!

0095	0.005 MHz / sec

BsaI...now down to reasonable forces...i think these three previous files prove that the sucrose is drastically increasing binding strength

0096	0.05 MHz / sec

BsaI in 1 m Sucrose

0097	0.005 Mhz / sec

BsaI in 1 m Sucrose

0098	0.5MHz / sec

BsaI in 1 m Sucrose

0099	0.5 MHz / sec

BsaI in 0.5 m Sucrose

0100	0.05 MHz / sec

BsaI in 0.5 m Sucrose

0101	0.005 MHz / sec

BsaI in 0.5 m Sucrose

I am now getting distressed that I am seeing too much weird shit...i think part of the stuff i have been seeing may have to do with multiple tethering?

0102	0.5 MHz / sec

BsaI in 0.25 m Sucrose

0103	0.05 MHz / sec

BsaI in 0.25 m Sucrose

0104	0.5 MHz / sec

BsaI in 0.125 m Sucrose

0105	0.05 MHz / sec

BsaI in 0.125 m Sucrose

0106	0.5 MHz / sec

BsaI in 0.0625 m Sucrose

0107	0.05 MHz / sec

BsaI in 0.0625 m Sucrose

0108	0.5 MHz, BsaI only (no sucrose)

0109	0.05 MHz, BsaI only (no sucrose)

010510.txt
5/10/01 3:31 AM



Step 0 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 1 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 2 :  Antidig from yesterday (20 ug / ml) ;  2.000000  s.v.;   5.000000  min.

Step 3 :  BGB 10 mg / ml (same tube at +4C for weeks) ;  2.000000  s.v.;   10.000000  min.

Step 4 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 5 :  17-mer, 1:5 dilution of freezer stock in PopBuf ;  1.000000  s.v.;   12.000000  min.

Step 6 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 7 :  Bangs 1:10 in PopBuf ;  1.000000  s.v.;   35.000000  min.

Step 8 :  PopBuf ;  10.000000  s.v.;   0.000000  min.

Step 9 :  see data notes for rest of protocol (BsaI and washes) ;  0.000000  s.v.;   0.000000  min.



1:05 AM flowed in 1:20 BsaI 2 s.v. in PopBuf

2:17 AM 5 s.v. popping buffer

2:27 another 5 s.v.

2:32 another 5 s.v.

3:25 AM another 5 s.v. after waiting this 1 hour

0111	0.5 MHz / s

Naked 17-mer

6 out of 14 (43% showed peeling) but only 3 / 14 (21%) showed complete peeling. Only 1 (7%) showed overstretch behavior

0112	0.5 Mhz / s

BsaI 1:20 popping buffer

First two segments STILL show overstretching. IDEA: Last night and tonight I have NOT turned off the room fan. This would lower the temperature of the sample, and perhaps affect the binding energy this significantly?

This overstretching went away after a few minutes and overall, I had lots of popping (very high percentage success). This is consistent with previous observations that fresh washes tend to revive good tethers.

0113	0.05 MHz / s

BsaI 1:20 popping buffer

4 segments, first and last show very nice low force popping

0114	1.85 V Force Clamped Pop

BsaI 1:20 popping buffer

segment is likely huge

0115	higher force than last file, still doesn't budge

0116	2.1 V force clamped

BsaI 1:20 popping buffer

higher force than last file, finally budges

this file has lots of great force clamp results

0117	2.2 V FC

BsaI 1:20 popping buffer

0118	0.05MHz/sec

BsaI 1:20 popping buffer

to show that there is still enzyme binding

0119	0.5Mhz / sec

BsaI after 5 s.v. wash

0120-0122, after 10 s.v., 15 s.v., 20 s.v.	0.5MHz / sec

0123	0.5Mhz / sec

After waiting an hour even...these guys just won't go away

0105102.txt
5/10/01 5:39 AM



Step 0 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 1 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 2 :  Antidig from yesterday (20 ug / ml) ;  2.000000  s.v.;   4.000000  min.

Step 3 :  BGB 10 mg / ml (same tube at +4C for weeks) ;  2.000000  s.v.;   12.000000  min.

Step 4 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 5 :  17-mer, 1:5 dilution of freezer stock in PopBuf ;  1.000000  s.v.;   11.000000  min.

Step 6 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 7 :  Bangs 1:10 in PopBuf ;  1.000000  s.v.;   25.000000  min.

Step 8 :  PopBuf ;  10.000000  s.v.;   0.000000  min.

Step 9 :  see data notes for rest of protocol (BsaI and washes) ;  0.000000  s.v.;   0.000000  min.



4:11 2 S.V. of BsaI 1:20,000 (this is the second so far)

4:19 AM, 2 s.v. BsaI 1:5,000

4:39 AM 2 s.v. BsaI 1:2,000

4:57 2 s.v. more of 1:2,000

5:05 2 s.v. 1:500

0124	0.5MHz / sec

BsaI 1:20,000, 2 s.v.

only 1 segment has pops

0125	0.05MHz / sec

BsaI 1:20,000 2 s.v. so far

0126	0.05MHz / sec

BsaI 1:20,000 4 s.v. so far

0127	0.05MHz / sec

BsaI 1:5,000 2 s.v.

0128	0.5MHz / sec

BsaI 1:5,000 2 s.v.

I am noticing a propensity to stick right at the END of template

0129	5 MHz / sec

BsaI 1:5,000 2 .s.v.

There are three complete peeler segments, which make me feel comfortable that nothing too freaky is going on (I hope)

0130	5 MHz / sec

BsaI 1:2,000 2 .s.v.

0131	0.5 MHz / sec

BsaI 1:2,000 2 .s.v.

0132	0.05 MHz / sec

BsaI 1:2,000 2 .s.v.

0133	0.05 MHz / sec

BsaI 1:2,000 2 .s.v. (second wash)

0134	0.5 Mhz / sec

BsaI 1:500 2 s.v.

0135	0.05 MHz / sec

BsaI 1:500 2 s.v.

010513.txt
5/13/01 6:24 AM



Step 0 :  Note, the sample was rather small, probably 80% of what i call s.v. below ;  0.000000  s.v.;   0.000000  min.

Step 1 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 2 :  PBS ;  5.000000  s.v.;   0.000000  min.

Step 3 :  antidig, thawed last week ;  2.000000  s.v.;   4.000000  min.

Step 4 :  10 mg / ml BGB (same as last week...is very old) ;  2.000000  s.v.;   1.250000  min.

Step 5 :  repeat above 2 times ;  0.000000  s.v.;   0.000000  min.

Step 6 :  17-mer, thawed and diluted 1:5 last week ;  1.000000  s.v.;   6.000000  min.

Step 7 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 8 :  Bangs 1:10 in PopBuf, diluted last week or earlier ;  1.000000  s.v.;   5.000000  min.

Step 9 :  repeat above once ;  0.000000  s.v.;   0.000000  min.

Step 10 :  Bangs 1:10 freshly into PopBuf ;  1.000000  s.v.;   5.000000  min.

Step 11 :  Repeat above once (a total of 4 bead washes) ;  0.000000  s.v.;   0.000000  min.

Step 12 :  PopBuf ;  10.000000  s.v.;   0.000000  min.

Step 13 :  BsaI 1:2,000 in PopBuf ;  1.000000  s.v.;   0.000000  min.



Second protocol:



Step 0 :  wash glass with water and kim wipes ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig 20 ug / ml freshly diluted ;  2.000000  s.v.;   9.000000  min.

Step 2 :  BGB 10 mg / ml ;  2.000000  s.v.;   9.000000  min.

Step 3 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 4 :  17-mer freshly diluted 1:5 ;  1.000000  s.v.;   8.000000  min.

Step 5 :  popBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs 1:10 PopBuf (fresh today) ;  1.000000  s.v.;   10.000000  min.

Step 7 :  repeat last step 1 time ;  0.000000  s.v.;   0.000000  min.

Step 8 :  PopBuf ;  10.000000  s.v.;   0.000000  min.

Step 9 :  BsaI, 1:2,000 in PopBuf ;  2.000000  s.v.;   0.000000  min.



5:23 AM BsaI 1:2,000 2 s.v.

0136	Naked DNA...the protocol did not produce very good tethers. Lots of non-specific jiggly beads...but I can find just enough peelers that I suppose it's worth trying the 1:2000 BsaI next file

0137	trying force clamp on naked DNA...abandoning sample after this

BEGIN NEW SAMPLE

0138

0139	Lots of popping...unfortunately, looks like I again have too much enzyme...or at least as time goes on?

0140	more of the same

0141	the first segment in this file is the same tether as last segment of last file. the popping is gone, but still pins at the same site

010704.txt
7/4/01 6:21 AM



Step 0 :  Begin with Bert's specially cleaned coverglass and slide, no further pre-rinsing ;  0.000000  s.v.;   0.000000  min.

Step 1 :  Antidig 20 ug / ml in Popping Buffer ;  1.000000  s.v.;   5.000000  min.

Step 2 :  BGB, 10 mg / ml in 50% popping buffer. Note, this BGB is maybe 1 or 2 months old at +4C...However, it has been left out at room temperature 2 or 3 times for over 24 hours each time. Oh well, I'm not going to remake it now. ; 5.000000  s.v.;   5.000000  min.

Step 3 :  PopBuff ;  5.000000  s.v.;   0.000000  min.

Step 4 :  New 17-mer peeling template, estimated 29 pM (Diluted 1:10 from stock into Popping Buffer) ;  1.000000  s.v.;   8.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs, freshly diluted 1:10 into popping buffer, and then sonicated (100 ul volume) for about 1 minutes by hand ;  1.000000  s.v.;   8.000000  min.



5:47 added BsaI, 5 s.v. 1:50 in PopBuf

Tethering stats:

stuck: N/A ; 19 ; 20 ; 16; 25

jiggle; 19 ; 17; 10; 9; 13 (see transient stickign)

0165	naked DNA, 0.05 MHz / sec

0166	0.05MHz / s

single tether, lots of data, forgot to increase the speed

(but is a double tether)

0167	0.5 MHz / s naked

SHould be several good peelers for comparison

0168	naked

peeled a long way, and then reversed. repeated same tether

0169	Bsai added...a reversal file

0170	BsaI added...quick reversals...a few good ones to examine

0171	BsaI, reversing before a pop (waiting 4,000 data points)

0172	some more like the previous

010705.txt
7/5/01 11:58 AM



Step 0 :  Begin with Bert's specially cleaned coverglass and slide ;  0.000000  s.v.;   0.000000  min.

Step 1 :  Antidig (same as two days ago) in Pop BUf ;  1.000000  s.v.;   5.000000  min.

Step 2 :  BGB (see notes from two days ago) ;  2.000000  s.v.;   5.000000  min.

Step 3 :  PopBUf ;  5.000000  s.v.;   0.000000  min.

Step 4 :  Today's Ligation reactions (either HindIII or XhoI), 1:2 into Pop Buf (unpurified ligation reaction) ;  1.000000  s.v.;   6.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs freshly 1:10 into PopBuf, sonicated for roughly 1 minute (100 ul volume) ;  1.000000  s.v.;   8.000000  min.

Step 7 :  PopBuf ;  10.000000  s.v.;   0.000000  min.



0173	0.5 MHz / s, HindIII cap

only 1 out of 18 segments showed the cap...not so good

0174	0.5 MHz / s, XhoI cap

Much better than HindIII cap...about 50% show capping (6 out of 13)

0175	0.5 Mhz / s XhoI cap, reversing at 0.05MHz / s

only first segment is good.

0176	0.5 Mhz / s XhoI...a couple good reversals

0177	0.5 Mhz / s HindIII...no good segments, really, but perhaps is a couple capped (but no reversal caps)

010710.txt
7/10/01 3:54 PM

Super-fast tethering protocol (no wait times: antidig; 5 s.v. BGB; 5 s.v. Pop; 1 s.v. DNA (30 pM, new 17-mer); 5 s.v. Pop; 1 s.v. Beads (about 3-5 minutes); 10 s.v. Pop, observe:

90 micron octagon fields:

stuck: 3;0;2;2;3;5;9;3;6;5

wiggle 0;2;0;2;5;5;7;1;0;3

added XhoI at 2:24 AM

197	trying to focus exactly on tether. This is producing stuck beads. This is either (A) because I changed the nomarski compenation and am now lower than previously tonight or (B) the sample is stickier due to the fast application of BGB...Actually, I just checked, and it seems that my eye sees "in focus" much higher when the compensation is set for zero (when the bead image is symmetric, as opposed to the usual 3-D thing). therefore, I will switch back to this method in next file

198	z=9.5, shows no evidence of sticking

199	still playing with z height

200	still playing with z height

201	z=10.25, first segment looks perfect

202	one peeler after adding fresh beads

203	0.5 MHz / sec Added XhoI in 1:50 Pop Buffer

Are there hints of force-mediated cutting?

204	0.05 MHz / s XhoI

0107102.txt
7/10/01 3:55 PM

tethering before adding any ligase buffer:

looking at TV screens (which are smaller than the actual video)

stuck: 0; 1; 0; 1; 1;1;0;0;1;0

wiggle: 3;0;3;1;3;4;3;2;1;2

Added 2 s.v. T4 DNA LIgase in LIgase buffer (no DNA, though) at 6:48 PM...Tethers did not stick immediately.\

TV screens:

stuck: 2;0;1;1;1;2;0;1;0;0

wiggle:2;1;0;3;1;2;5;5;1

7PM added all components

8PM added: 2 s.v. (1 ul T4 DNA ligase--progema; 2 ul 10x Ligase buffer; 2 ul pCP681 EarI sticky from Jan. 27, 2001; 15 ul h2o)

11 PM, flowed 10 s.v. of 1x Ligase BUffer (Promega) through what I'll call sample "B" (made at the same time as A, but haven't added anything after the bead wash at 7PM). File 190 will be a pre-ligation reaction control in the 1x Ligase buffer

12:25 AM flowed more beads into sample A and washed out after 10 minutes

178	anchoring

179	Added s.v. T4 DNA ligase (no DNA) in 1x Ligase buffer and also 0.02% Tween-20.

This file should have good statistics for control (don't really see much difference, except 20% of tethers may have higher shearing / peeling force)

180	added complete reaction at 7PM...this file shows no peeling in the first 5 minutes

181	no peeling after 10 minutes

182	no peeling after 15 minutes

183	no peeling after 1/2 hour

184	no peeling after 45 minutes...i give up

185	still no peeling

186	first 5 minutes after adding second reaction (8PM)

shows two peelers

187	10 minutes after reaction...no peelers

188	?

189	10 PM, having left reaction going for 2 hours. See 2 complete peelers out of 24 segments, for about 8% efficiency. Not great, but not terrible.

190	control sample B

191	Added P18-cap-High LIgation reaction..no clear peelers

192	After about 25 minutes, there is still no good evidence of successful ligation (even though cap concentration is extremely high (10 micromolar)

193	Some more of the same

195	Took 15 segments back in Sample A, and there were NO peelers. THis is pathetic

196	Sample A, after adding fresh beads, and in fact getting plenty of loose tethers. Only the last segmetn (out of 16) peels, showing pitiful ligation efficiency. There could be something wrong with the method

010710_2.txt
7/10/01 9:04 PM

Used Richard's coverglass, and tethering is not at all as good.

about 2 or 3 times as many stuck beads as wigglers. about 5 wigglers per 90 micron octagon

4:24 PM, XhoI 1:200 in Pop Buff, 2 s.v.

205	Pre-XhoI, 0.5 MHz / sec, 30 tethers

206	XhoI, 0.5MHz / s, 30 tethers

207	XhoI, 5 MHz / s, 30 tethers

208	XhoI, 0.005 Mhz / s (VERY SLOW) 18 tethers

Watching the oscilloscope, I notice that at the very beginning, there are very many kinds of flip-flopping events (time scales and length scales) it would be interesting to take non-decimated early data...

209	XhoI, 0.05 Mhz / s 30 tethers

I would say sample is degrading

210

211	XhoI, after washing many sample volumes(15 to 20) with popping buffer...lots of breaking, probably no popping

212	XhoI washed, 0.5MHz / s 10 segments

After having washed with 15 s.v. of 1 mM Tris + 0.01% Tween, and the 5 s.v. PopBuf

There is still evidence of popping at the early non-specific site, and things are still breaking

213	XhoI washed with many buffers (finally with 2 M NaCl), 0.5 MHz / s, 30 segments

010711.txt
7/11/01 5:40 AM



Step 0 :  Start with Karen's (stolen from Richard, cleaned by Brent) slides...not so clean ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig 20 ug / ml in Pop Buf ;  1.000000  s.v.;   3.000000  min.

Step 2 :  BGB ;  2.000000  s.v.;   7.000000  min.

Step 3 :  Pop Buf ;  5.000000  s.v.;   0.000000  min.

Step 4 :  New 17-mer, 1:2 (already 1:10 earlier) in Pop Buf, estimated 15 pM ;  1.000000  s.v.;   3.000000  min.

Step 5 :  Pop Buf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs 1:10 Pop Buf, sonicated earlier today ;  1.000000  s.v.;   10.000000  min.

Step 7 :  Pop Buf ;  10.000000  s.v.;   0.000000  min.

Step 8 :  One sample only (for now) BamHI, 1:200 in Pop Buf (Life Tech) ;  0.000000  s.v.;   0.000000  min.

Step 9 :  Stretch For a While ;  0.000000  s.v.;   0.000000  min.

Step 10 :  Other Sample, EcoRV at about 4:34 AM (1:50 in PopBUf) ;  5.000000  s.v.;   0.000000  min.



Added Eco RI

214	0.5 MHz / s A few BamHI files...NOTEThere are extra sites!!!

215	0.5 MHz / s

216	0.5 MHz / s in 10 mM CaCl2

EXCEPT AT END! FORGOT TO SWITCH FILES!

217	EcoRV i nPop Buf 0.5 MHz / s

Maybe a couple non-specific bindings here and there, but for the most part, either non-detectable sliding, or no binding

218	EcoRV 5 MHz / s

219	EcoRV 0.05 MHz / s

220	EcoRV Calcium + PBS 0.05 MHz / s

1 segment only

221	EcoRV Calcium 10 mM in PBS 0.5 MHz / s

Sample is completely fucked up (precipitate) all beads stuck in a matter of minutes. Maybe got one pop

010712.txt
7/12/01 4:55 PM



Step 0 :  Bert's slides ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, fresh, 1:10 in PopBUf (20 ug / ml) ;  1.000000  s.v.;   3.000000  min.

Step 2 :  BGB 10 mg / ml,  ;  2.000000  s.v.;   10.000000  min.

Step 3 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 4 :  17-mer, 1:4 in PopBuf ;  1.000000  s.v.;   6.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs, soncicated, 1:10 PopBUf ;  1.000000  s.v.;   12.000000  min.

Step 7 :  PopBuf ;  10.000000  s.v.;   0.000000  min.

Step 8 :  BsoBI (one sample only), 1:50 in PopBuf ;  2.000000  s.v.;   0.000000  min.



3:57 PM 1:10,000 BsoBI in Sample B

4:40 PM Washed with 5 s.v. PopBUf

222	BsoBI 0.5 MHz/ s

Just a couple files, shows popping

223	BsoBI 0.005 MHz / s

A single segment with peeling

224	BsoBI 0.05 MHz / s

Segment 8 possibly shows sliding

225	BsoBI 0.5 MHz / s, 1:10,000

226	BsoBI 5 MHz / s 1:10,000

30 segments

227	forgot to check reversal box

228	BsoBI 0.5 MHz / s, stop after 4000 points, reversal

Same tether 4 times

229	BsaBI 0.5 MHz / s, washed 5 s.v. (sample A from before)

Looks like enzymes diss. very quickly?

230	BsaBI 0.5 MHz / s sample A, washed 5 times (same as last file)

Shows some zero poppers, but also many poppers

010713.txt
7/13/01 10:58 AM

This sample is for studying z-height (will do many z telescope settings)



Step 0 :  Karen's Slides & cover ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, fresh 1:10 popBuf ;  1.000000  s.v.;   3.000000  min.

Step 2 :  BGB (I left it at room temperature AGAIN!...probably like 6 hours) ;  2.000000  s.v.;   8.000000  min.

Step 3 :  New 17-mer (1:4 dilution of previous 1:10 dilution...was also at room temperature last night) (Forgot to wash before this step) ;  1.000000  s.v.;   3.000000  min.

Step 4 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 5 :  Bangs 1:10, freshly in popBuf, sonicated 1 minute ;  1.000000  s.v.;   10.000000  min.

Step 6 :  PopBuf ;  10.000000  s.v.;   0.000000  min.

Step 7 :  Seal ;  0.000000  s.v.;   0.000000  min.



With z = 8.5, it seems like I have to focus about 500 nm +/- 200 nm into solution in order to make reflection of laser beam look small. This would imply that trapping center is about 500 nm above beam waist?

Arbitrary estimation of when beam waist hits surface, when I am focused on surface (multiple measurements, but will undoubtedly be biased): 10.55 mm; OFF-SCALE...HMMMMM  I was "wrong" on the first measurement (or at least I changed my mind as to how it should look when the beam hits the surface of coverglass). Video on Koch VII, 9 AM today...shows dust and laser, with z = (offscale positive, estimated 13.5 mm)

Laser = 1V, 14 amps. Estimation of parfocal bead: 9.35; 9.14; 9.13; 8.95; 9.36; 9.08; 9.19; 9.25; 9.01; 9.34 (9.18 +/- 0.144 mm) fairly insensitive to depth into solution, but these measurements were taken close to surface, but far enough away so that trapped bead could drop below focus without hitting surface.

Laser = 4V, 14 amps. Adjusted DIC. parfocal bead: 9.08; 9.14; 9.34; 9.14; (refocused objective) 9.18; 8.99; 9.02; 9.18; 8.90; 9.18 (avg = 9.12 + / - .123 mm)

Laser = 4V, focusing on small dust particles, trapped free bead. Lower z telescope, until I think bead is stuck (and I am right, because it is stuck); Therefore, this is a lower limit for when a trapped bead sticks, when focused on the surface. Multiple measurements: 6.95 mm; 8.95 mm; [start to notice change in signal at 10.25 mm] 9.20; 9.38; 9.92

[Note, this gives an estimate of dust focusing error]

Same as last, but now am just stepping the z-telescope down, so I can get an upper and lower bound...And I am saying it is not stuck if it flies out of trap momentarily when I cycle beam off / on quickly. multiple measurements: 9.1 to 9.2 mm; 9.9 to 10.0 mm; 9.7 to 9.8 mm; 9.5 to 9.6

[Note, this gives an estimate of laser focusing method error]

Same as last, but am using laser pattern at z = 10.5 mm to focus on surface. 8.6 to 8.7 mm; 8.8mm to 8.9 mm; [VIDEO KOCH VII 9:54 AM, focusing method; bead flew away at 9.3 mm, though]; 8.9 mm to 9.0 mm; 9.5 mm to 10 mm; 9.0 to 9.1 mm

230	z = 11 mm

DO NOT LIKE THIS FOCUSING METHOD...WILL ABANDON

231	z = 13 mm

Focusing on laser beam, as discussed in journal to data file (calibrated image at z = 13 mm, and very small dust particle...that is, a DIM dust particle)

232	z = 12 mm

233	z = 11 mm

234	z = 10.25 mm

not even sure if i got a single full peeler, but this method is much too much of a pain in the ass.

010713_2.txt
7/13/01 1:41 PM



Step 0 :  Karen's Slides & cover ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, fresh 1:10 popBuf ;  1.000000  s.v.;   3.000000  min.

Step 2 :  BGB (I left it at room temperature AGAIN!...probably like 6 hours) ;  2.000000  s.v.;   3.000000  min.

Step 3 :  BGB ;  5.000000  s.v.;   0.000000  min.

Step 4 :  New 17-mer (no dilution of previous 1:10 dilution...was also at room temperature last night) (Forgot to wash before this step) ;  1.000000  s.v.;   3.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs 1:10, freshly in popBuf, sonicated 1 minute ;  1.000000  s.v.;   10.000000  min.

Step 7 :  PopBuf, supplemented with 1mM biotin, which also results in 1% DMSO ;  10.000000  s.v.;   0.000000  min.



PLEASE NOTE THE BIOTIN! (And DMSO)

Washed again with popping buffer, because tethers were falling off surface when i looked after step 7. This was probably due to DMSO, in my opinion.

At 1:33 PM Added BsoBI 1:2,500 in:

10% PopBuf in addition to:

10 mM EDTA (so, final is 11 mM)

0.02% Tween (so, final is 0.022%)

235	z = 10.25

This can go with the last group of files...I replaced the DMSO buffer with popping buffer, so now should be the same peeling forces.

236	high data rate, for early fluctuations

237	?

238	One peeler after adding the BsoBI...the only one i could find. All tethers either stuck or detached, i cannot tell which. Unexpected result of the low-salt buffer.

010714.txt
7/14/01 6:47 PM

V:\Aatte\koch\data\Popping_VC_BsoBI



Step 0 :  Karen's Slides & cover ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, yesterday 1:10 popBuf ;  1.000000  s.v.;   1.000000  min.

Step 2 :  BGB same tube ;  5.000000  s.v.;   2.000000  min.

Step 3 :  BGB ;  5.000000  s.v.;   0.000000  min.

Step 4 :  New 17-mer (no dilution of previous 1:10 dilution.) ;  1.000000  s.v.;   2.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs 1:10, freshly in popBuf, sonicated 1 minute ;  1.000000  s.v.;   8.000000  min.

Step 7 :  PopBuf, supplemented with 1mM biotin, which also results in 1% DMSO ;  5.000000  s.v.;   0.500000  min.

Step 8 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 9 :  BsoBI, very roughly 1:100 in PopBuf, added at about 5:30 PM, at least 10 minutes before first stretching ;  2.000000  s.v.;   0.000000  min.



About 5 tethers per 90 micron octagon, and about 10 stuck beads

DataFile003: Washed with 5 s.v. PopBuf (no Enzyme at 6:27 PM)

002	0.5 MHz / s; 50 segments

003	0.5 MHz / s; 30-ish segments in no-enzyme buffer. The purpose of this file is to get information about what kinds of things happen during dissociation. For example:

A. Do certain sites dissociate earlier?

B. What is the kind of rate?

C. Do the enzymes seem to dissociate from the ends of the template?

010715.txt
7/15/01 4:16 PM

V:\Aatte\koch\data\Popping_VC_BsoBI



Step 0 :  Karen's Slides & cover ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, yesterday 1:10 popBuf ;  1.000000  s.v.;   1.000000  min.

Step 2 :  BGB same tube ;  5.000000  s.v.;   1.000000  min.

Step 3 :  BGB ;  5.000000  s.v.;   0.000000  min.

Step 4 :  New 17-mer (no dilution of previous 1:10 dilution.) ;  1.000000  s.v.;   2.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs 1:10, freshly in popBuf, sonicated 1 minute ;  1.000000  s.v.;   4.000000  min.

Step 7 :  PopBuf, supplemented with 1mM biotin, which also results in 1% DMSO ;  5.000000  s.v.;   0.500000  min.

Step 8 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 9 :  BsoBI, 1:100 in PopBuf, added at about 5:30 PM, at least 10 minutes before first stretching ;  2.000000  s.v.;   0.000000  min.



Much fewer beads than yesterday...probably about 1 tether per octagon at most

0004.dat	5 MHz / s, 50 tethers

010719.txt
7/19/01 4:28 AM

V:\Aatte\koch\data\Popping_VC_BsoBI



Step 0 :  Karen's Slides & cover ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, fresh 1:10 popBuf ;  1.000000  s.v.;   1.000000  min.

Step 2 :  BGB same tube ;  5.000000  s.v.;   1.000000  min.

Step 3 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 4 :  New 17-mer (1:4 dilution of previous 1:10 dilution.) ;  1.000000  s.v.;   2.000000  min.

Step 5 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 6 :  Bangs 1:10, diluted fresh in popBuf, sonicated 1 minute today ;  1.000000  s.v.;   5.000000  min.

Step 7 :  PopBuf, supplemented with 1mM biotin, which also results in 1% DMSO ;  5.000000  s.v.;   0.500000  min.

Step 8 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 9 :  BsoBI, 1:100 in 90% popbuf, 400 m Sucrose ;  2.000000  s.v.;   0.000000  min.



Tethering not so great...difficult to find loose tethers

0006	0.05 MHz / s; 400 m sucrose

0007	0.05 MHz / s; 10 mM Tris only (no enzyme, but for what remained during / after wash)

0008	0.05 MHz / s CalTris (50 mM CaCl, etc.)

MOst of the tethers stuck (99% ish)

A couple peelers clearly show higher peeling force...certainly than the 10 mM Tris sample

010719_2.txt
7/19/01 8:53 AM

V:\Aatte\koch\data\Popping_VC_BsoBI



Step 0 :  Karen's Slides & cover ;  0.000000  s.v.;   0.000000  min.

Step 1 :  antidig, fresh 1:10 popBuf ;  1.000000  s.v.;   4.000000  min.

Step 2 :  BGB same tube ;  5.000000  s.v.;   1.000000  min.

Step 3 :  New 17-mer (1:4 dilution of previous 1:10 dilution.) ;  1.000000  s.v.;   4.000000  min.

Step 4 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 5 :  Bangs 1:10, diluted fresh in cal5Tris, sonicated 1 minute today ;  1.000000  s.v.;   7.000000  min.

Step 6 :  Cal5Tris, supplemented with 1mM biotin, which also results in 0.5% DMSO ;  5.000000  s.v.;   0.500000  min.

Step 7 :  Cal5Tris ;  5.000000  s.v.;   0.000000  min.

Step 8 :  BsoBI, 1:100 in Cal5Tris ;  2.000000  s.v.;   0.000000  min.



Tethering actually looks pretty good.

0009	0.05MHz / s, auotJ, cal5Tris BsoBI

FORGOT TO SET ALL OF THE AUTOJ PARAMETERS!

It is possible that slide was not firmly attached

0010	0.5 MHz / s, autoJ, cal5Tris BsoBI

30 segments

It is possible that slide was not firmly attached

0011	2 MHz / s autoJ, cal5Tris BsoBI

31 segments

It is possible that slide was not firmly attached

0012	0.2MHz/s autoJ, cal5Tris BsoBI

30 segments (I fixed slide, I think...if there were a problem before at all, which i don't really know)

0013	0.05 MHz / s autoJ, cal5Tris BsoBI

25 segmetns

010829.txt
8/29/01 10:59 PM



Step 0 :  This protocol is inexact, but pretty close to the usual ;  0.000000  s.v.;   0.000000  min.

Step 1 :  Bert's glass & Karen's slide cover, rinsed with water ;  0.000000  s.v.;   0.000000  min.

Step 2 :  antidig, from last week 1:10 PBS ;  1.000000  s.v.;   4.000000  min.

Step 3 :  BGB:  from a couple weeks ago: 20 mg / ml in H20 and then diluting 1:2 into PBS, then adding 4 M NaCl to 100 mM. I thin k the BGB precipitated, because the solution was cloudy, and it would not go through the 0.2 micron filter. I used this anyway. ; 2.000000  s.v.;   4.000000  min.

Step 4 :  BGB ;  2.000000  s.v.;   5.000000  min.

Step 5 :  BGB ;  2.000000  s.v.;   0.500000  min.

Step 6 :  New 17-mer, 1:20 dilution of the rest of the gel extraction from the first week of July ;  1.000000  s.v.;   6.000000  min.

Step 7 :  Bangs 1:10, diluted fresh in popBuf, sonicated 1 minute today ;  1.000000  s.v.;   8.000000  min.

Step 8 :  PopBuf, supplemented with 1mM biotin, which also results in 1% DMSO ;  5.000000  s.v.;   0.500000  min.

Step 9 :  PopBuf ;  5.000000  s.v.;   0.000000  min.

Step 10 :  EcoRI, from 10x Concentrated, 1:100 into PopBuf (100 ul) added at 10:30 PM ;  5.000000  s.v.;   0.000000  min.



Added the EcoRI (Concentrated, 1:100) at about 10:30 and sealed

0001	Strange results

0002	Strange results

011119.txt
11/19/01 11:19 PM

Short Capped Template Unzipping

NOTE: Even though this slide has VERY low tether density, the ratio of stuck to tethered is also very low. I would put the ratio at about 1:1, with about 1/4 tether per octagon (or maybe less).

So, something about Karen's blocking method is in fact very successful. Perhaps it is because I have incubated the beads for sometime in BGB buffer.

0001	Low cap template, 0.1 Mhz / s

0002	Low cap template, 0.01 MHz / s

0003	Again, but later on. There may be a couple interesting data sets here, but overall the capping is not working at all.

011201.txt
12/1/01 3:34 PM

Short Capped Template Unzipping

Low tether density again!

"Internal / Low" sample:

stuck / tether: 4/0; 4/0; 6/0

Many fewer tethers than "high" sample...I would say about 1 per five 200 micron fields

"Internal / High" sample:

stuck / tether: 6/ 1; 4/0; 5/0

It is overall pretty easy to find tethers, probably about 1 per 200 um field

0004	High capped template

0005	Low Capped template

0006	repeating a tether (with reversal)

0007	Low cap, reversal still on

011203.txt
12/3/01 10:35 PM

Today will be normal uncapped unzipping template (not gel extracted).

First I am comparing heated and unheated samples

Files will be stored in Popping_VC_Osmolite\

Heated sample (stuck / tether): 4 / 0; 0 / 0; 4 / 0; 0 / 0; (don't see any tethers in like 10 full-fields)

non-heated sample: 3 / 0; 1 / 0; 2 / 0; 3/ 0; 3 / 0



THERE ARE NO TETHERS IN EITHER SAMPLE

011203_2.txt
12/3/01 11:18 PM

This is trying tethering (according to the usual recent protocol) with the very diluted 17-mer from July. There are still NO tethers (I stretch the 3 I could find that perhaps looked like tethers, but no unzipping).

I wonder if something is truly fucked up with my buffers or other supplies?

0001	No tethers

011204.txt
12/4/01 10:03 PM

These are the end-labeled templates. At first I am hoping to see a very good tethering efficiency, given what I have learned lately about the failure of the internal reactions lately.

Yay!

This is a great control (unfortunately, somewhat). First, I see about 1 tether per 90 micron octagon in the bottom sample (high-cap, I think), and I see about 0.5 per 90 micron field in the top sample (low-cap, I think)

Now for stretching.

0008	top sample (low I think)

(With reversal)

0009	same sample (top), with reversal turned off

0010	bottom sample

011204_3.txt
12/4/01 12:28 AM

Trying another sample, with:

New antidig

New PopBuf

Beads are not in blocker but are just in popbuf

None of the biotin quenching

17-mer from July

And, I see NO tethers. Well, actually I see about 1 or less per big (200 um) field...I stretched about 7 of them, and only saw unzipping twice.

So, I mixed 10 ul of beads with some PCR-tagged DNA that has been sitting in the fridge for MONTHS...I flowed in, and tethers started forming IMMEDIATELY. Huh.

Files are in Fluctuations/Overstretch/

PoppingVC_osmolite/0002	No tethers

Fluctuations\Overstretch\0001	Some overstretch

Fluctuations\Overstretch\0001	Some overstretch

011214.txt
12/14/01 1:07 AM

This is the new pBR322 construct.

The protocol can be found in \PaperI\EQ constant\011213\*.ini

There are VERY FEW tethers. But I could easily find a few. There are less than 1 tether per 10 90 micron fields, depending on whether the slight jigglers are tethers. There are about 2 stuck beads per 90 micron

Flowed in cold 10xEcoRI 1:10 in Kk Buffer at 11:55 PM.

After taking EcoRI data, I flowed in old beads in BGB popping buffer, and then washed away after about 10 minutes (1:05 PM). I did not see a significant increase in tethers, which may lead one to believe that there is little DNA on the surface, or that it is laying down stuck.

0005	naked pBR322

0006	naked pBR322

0007	EcoRI, 0.5 MHz / sec

0008	EcoRI, 0.3 MHz / sec, with proportional to 300

0009	EcoRI, 0.1 MHz / sec, with proportional to 300

011214_2.txt
12/14/01 4:12 AM

THis is the second sample of the night.

I put in 10 times as much DNA, but there seems to be even FEWER tethers! Argghhh!

Enzyme added at 3:40 AM.

0010-0012	10xEcoRI 1:1000, 0.1 MHz / s proportion v-clamp referenced to 100

020122.txt
1/22/02 3:21 PM

Using the popping buffer tethering protocol...the tethering efficiency is much better than when i tried the Kk buffer last month.

2:12 PM, added EcoRI (10x), 1:1000 in popping buffer + ~10 nM hindIII capping oligo

2:33 PM...I found a tether farm in the middle of my sample!!! tether density increased from about 1 to 50 tethers per octagon

Popping_VC_EcoRI\020122\0013	Naked DNA

0014-0015	Data files for Richard

0016-0019, for measuring EQ constant of EcoRI in popping buffer on pBR322 site	Files 0017 & 18, I am in what is called the "tether farm"...

File, 20, 21	pre-shining laser onto tether before stretching

FIle 22	Being VERY careful not to pre-shine laser before stretching (and thus centering is pretty bad)

020122_2.txt
1/22/02 10:30 PM

About 9:09 PM, added EcoRI 1:2,500 (10x stock) in Kk buffer (+ ~ equimolar companion = 3:2,500 of 1:80 dilution of P15 / P17 duplex, which has EcoRI site). The tethering protocol was the popping variety, but I flowed 4 sample volumes of Kk buffer through before adding EcoRI. I will start taking data immediately after adding EcoRI.

For the second batch of data (0026 ->), I added EcoRI, 10x 1:2500 in Kk buffer plus 100 mM extra NaCl, plus 1:10 of 5 mg / ml BGB in Hepes. The dilution was performed 1:50 twice.

before 0026	first batch of data

0026	directly after adding the second batch of (high salt) ecoRI

020123.txt
1/23/02 11:44 PM

Eco added at 10:08:30

Tethering protocol is the same as the popping buffer protocol from yesterday. I did not switch to the Kk buffer until I added the EcoRI, at which point, I flowed in 4 sample volumes of the EcoRI (I think it was 1:800 instead!!)

10:46 PM, added the following (1:400)

1:20 was 1 ul Eco added to 16.6 ul Kk plus 0.4 ul 5 M NaCl plus 2 ul 5 mg / ml BGB in Hepes

1:400 was 1 ul of the 1:20 added to 35.2 ul Kk plus 0.8 ul 5M NaCl plus 4 ul 5 mg / ml BGB in Hepes (NO!  I think I screwed up the second dilution and used 1:800 instead!)

An important note with the second set of samples (with BGB), is that I am moving towards the entrance of the sample as time goes on. It seems like the popping frequency is increasing with time, but it's hard to say first whether that's even true and second, whether it's due to time or spatial...

Added the 150 mM NaCl at 11:18:40 PM. This one was made by diluting:

2 ul EcoRI 10x stock

0.536 5 M NaCl

4 ul BGB

33.5 ul Kk buffer

And then repeating this 1:20 dilution. I expect there to be > 50% occupancy.

NOTE: Above I was actually using 1:800 dilution!!!

Sheesh, the new 1:400 in 150 mM salt had TONS of occupancy. I wonder if now it's going to be too high of a Ka?

0029	One naked tether

0030	9 tethers, directly after adding the EcoRI

0031	After giving the sample a bit more time to equilibrate

<=0035	THese are with the BGB sample

0036	150 mM NaCl (this is more exact than the previous) plus 0.5 mg / ml BG in Kk derived buffer

020124.txt
1/24/02 6:18 PM

Typical popping buffer tethering protocol, following which, I added

EcoRI 1:80 into Kk based 250 mM NaCl buffer

Before adding the EcoRI, there are plenty of tethers, at least two per 90 micron

Added the EcoRI at about 4:40 PM

Flowed in 4 more s.v. of 1:80 EcoRI at 4:46:15 PM...I saw very low popping before, compared with expected. After a few more data sets, I will in fact up the stretch rate, because maybe the expected popping force is a bit too low?

At 5:15:15 PM, added 225 mM Kk based 1:80 EcoRI

NOTE: I think I may have used the WRONG BGB for the last sample (that is, popping based, instead of Hepes based). Therefore, I will repeat.

Added the repeat with the correct Hepes BGB at 5:32 PM

6:04 PM, added the 165 mM sample (1:400)...this sample looked a lot better

0040	250 mM NaCl, saw very little popping, possibly too early???

0046	225 mM NaCl

0048	New batch of 225 mM

after 6:04 PM...the 165 mM sample

020125.txt
1/25/02 4:14 PM

Repeating the 225 mM Data point, in hopes that the higher stretch rate will reveal more popping.

Added the enzyme at 3:03:30 ish

3:37:30 ish, added the 125 mM sample

4:01:30 ish, added the 100 mM sample

0054-0056	225 mM NaCl in Kk + 0.5 mg / ml BGB 10 MHz / s

0057-0059	125 mM NaCl in Kk + 0.5 mg / ml BGB 10 MHz / s

0060-0062	100 mM NaCl in Kk + 0.5 mg / ml BGB 10 MHz / s

020127.txt
1/27/02 8:20 PM

Popping buffer tethering protocol with 17-mer from July.

Then adding EcoRI 1:100 in 150 mM Kk buffer + 500 ug / ml BGB

Trying to do close site popping first, and then perhaps also complete template mapping

Good tethering before EcoRI, about 1 stuck and 1 tether per 90 micron octagon

EcoRI flowed in about 6:19 PM

Flowed in 5 s.v. more of the EcoRI at 6:56 PM

0063-0065	1 MHz / s 10 decimation

0066-0068	0.2 MHz / s 10 decimation

020127_2.txt
1/27/02 8:59 PM

New sample at roughly 8:30PM.

THis is using a 1:10 dilution of the "internal biotin / 17-mer / hindIII capped" unzipping construct from December 05, 2001 (which I have minimal notes on in my notebook). I am guessing it's about 200 pM stock concentration. The tethering protocol is the same as with the others lately: popping buffer protocol.

FLowed in EcoRI at about 8:37:30

Hmmm...somehow my sample turned really ugly after flowing in EcoRI. I don't know what happened. Perhaps the 150 mM NaCl is bad for the 17-mer? (Doubt it)

Perhaps I got immersion oil into the sample? That is possible, since I had too much on the condenser side.

0069	0.2 MHz / s 10 decimation; new sample; naked DNA

0070	0.2 MHz / s 10 decimation; new sample; 1:400 EcoRI

bubble in oil

0071	got rid of bubble

020128.txt
1/28/02 1:01 AM

New sample at roughly 11:45 PM

THis is using a 1:10 dilution of the "internal biotin / 17-mer / hindIII capped" unzipping construct from December 05, 2001 (which I have minimal notes on in my notebook). I am guessing it's about 200 pM stock concentration. The tethering protocol is the same as with the others lately: popping buffer protocol.

THis time, I got rid of the biotin quenching step

11:51 PM Sample did not have enough tethers

12:35PM Very, very few tethers in the 1:10 end-biotin sample

AND, there aren't any tethers in the undiluted internal biotin sample

There must be some other sort of tethering problem (dig or biotin?)

Tried flowing in freshly diluted beads in BGB, and no more tethers formed

0073	0.2 MHz / s 10 dec; naked 17-mer

0074	End-biotin version

020128_2.txt
1/28/02 3:52 PM

1:5 dilution of the internal anchoring 17-mer I tried yesterday. I added 200 ug / ml to the stock DNA tube. Also, I am using fresh antigid and fresh beads. I deleted the biotin quench step.

Tethers are about a couple per 90 micron ocatagon. THere are more stuck beads then yesterday. I stretch 9 naked tethers (file 75) and got 2 good stretches. Lots of weird shit happens. I wonder if I'm too close to the surface?

About 2:50 PM, flowed in 1:400 EcoRI in 125 mM NaCl...I saw beads going by the screeen as I flowed.

At 3:23 PM, changed to 1:3600 EcoRI in 125 mM NaCl by flowing in 6 s.v. of the new concentration. I will also switch back to 2 MHz / s

Indeed, the 1:3600 sample looked a lot better...I may have even gotten a good Figure 3

0075	naked DNA

0076	naked DNA, focusing intentionally high. I don't think the strangeness is reduced at all.

0077	A few stretches at 0.2 MHz / s

0078	Deciding to switch to 0.1 MHz / s

0080-0083	1:3600 EcoRI, 2 MHz / s

020129.txt
1/29/02 2:51 PM

Flowed in EcoRI 1:3600 in Kk 125 mM + 0.5 mg / ml BGB at about 2:21:30PM

0084	naked pBR322 in poppping buffer

0085-0090	125 mM EcoRI pBR322, 0.5 MHz / s VC

020129_2.txt
1/29/02 8:59 PM

Trying two new pBR322 samples, both 1:10 dilutions, but one I sonicated for about 30 seconds after dilution.

sonicated: stuck / tether:  2/2; 0/1; 0/1; 0/3; 0/2  (overall, looks pretty nice)

no sonicated: s / t: 2/7; 0/1; 3/1; 0/ 2; 2/1 (overall, can't say is different than sonicated sample)

Added EcoRI at about 8:15 PM

0090	Naked DNA

0091-0094	EcoRI 1:3600 125 mM pBR322 1MHz / s (about 10 pops)

0095-0097	same, but 2 MHz / s (about 11-13 pops...some overstretch)

0098-0100	same, but 1.5 MHz / s

020131.txt
1/31/02 5:48 PM

New capped 17-mer; Popping based protocol

Added 1:3600 EcoRI (125 mM Kk + 0.5 BGB) at about 5:34:30 PM

Before 0106	Various conditions of the new Proportional (J&F) clamp on naked 17-mer

106	With EcoRI; F & J enabled

107	EcoRI, F only

020206.txt
2/6/02 11:26 PM

Popping based tethering of pBR322. Antidig and Beads were diluted at least 1 week ago.

Then adding EcoRI at 11:01 PM, 1:3600 in 150 mM

0109?-0113	All EcoRI 1:3600 in 150 mM Kk

020207.txt
2/7/02 11:42 PM

Popping based tethering of pBR322. Antidig and Beads were diluted at least 1 week ago.

Then adding EcoRI at 10:45:30 PM, 1:6400 in 125 mM NaCl Kk

There is a weird spot in the Bertrand lens, somewhere between the condenser iris and the polarizer / 1/4 wave plate i think.

Flowed in EcoRI 1:50 in 250 mM NaCl at 11:15 PM

0113-0116	All EcoRI 1:6400 in 125 mM Kk

0117-0122	EcoRI 1:50 in 250 mM Kk

020209.txt
2/9/02 4:54 PM

Popping based tethering of pBR322. Antidig was diluted at least 1 week ago.

Beads are fresh dilution in 5 mg / ml BGB today

Then adding EcoRI at 3:40:30 PM, 1:4900 in 100 mM NaCl Kk (1x Kk plus 500 ug / ml BGB) (7 s.v.)

Added 6 s.v. 1:3600 EcoRI in 205 mM NaCl at 4:13PM, after washing with 5 s.v. of Popping Buffer.

Added 4 s.v. of 1:400 EcoRI at 4:32:30 PM (the above conc. was a mistake)

0123-0126	100 mM; 1:4900

0127-0130	205 mM 1:400

020301.txt
3/1/02 2:57 PM

Tethering is popping based with pBR322 1:10. There are a lot of tethers before adding EcoRI.

EcoRI added (No Ca or EDTA, "125 mM" NaCl) @ 1:31:30 PM

@1:57 PM, washed with 10 s.v. of 1x Kk Metal buffer

2:29:30 PM, added same EcoRI as above, except with 92 micromolar of CaCl2 added (so, EcoRI is 1:484 in 125 mM NaCl, 0.092 mM CaCl2)

0131	125 mM NaCl 0 Ca

0.1 MHz / s, jref = 600; Fref = 15 pN

0132-0133	Same as last, except changing initial (pre) stretch rate lower, to prevent overstretch

0134-0136	EcoRI plus Ca, stretch rate reduced to 0.025

020305.txt
3/5/02 2:14 PM

Tethering is popping based with pBR322 1:20. (Difference in protocol is that instead of washing beads away with 5 mg / ml BGB in popping buffer, I washed with 0.5x Kk Metal Buffer) Tethering is sort of crappy (moderate amount of stuck, very non-uniform tether density)

0137-0140	EcoRI 0.025 MHz / s (prop F & J) 0 mM Ca 125 mM NaCl

020308.txt
3/8/02 4:11 PM

With this new washing procedure (windex), stuck beads are not too plentiful. Seems to be comparable number of tethers and stuck beads. I haven't looked around yet to see whether there are still tether farms. After looking around a bit more, still no tether farms. I would say about 1 tether / 90 micron, and fewer than that for stuck beads.

After a while (3:56 PM), I did find a small tether patch (about 20 microns y dimension, 175 microns wide (x)).

At 4:01 PM, found a much bigger tether farm. There is a tall piece of gunk (say 20 microns diameter, infinitely tall), and in the patch around it, there is a tether farm about 100 microns in y dimension, and several hundred microns wide.

Another trend I seem to notice is that the tethers seem to like to form near or on bumps. I notice this, because I do not stretch tethers that have visible gunk associated with their area of the slide (where I think the bead is interacting with the gunk).

0141-0144	EcoRI 1:484 in 1x Kk Buffer "125 mM" NaCl. 100 kHz / s j600 F15

020309.txt
3/9/02 1:05 AM

@ 10:38:30PM, I added the EcoRI in 0 mM Ca 0 mM EDTA "125" mM NaCl.

Protocol is same as earlier today (windex wiped glass). Following beads, I washed with 2 s.v. 5 mg / ml BGB in Popping buffer, followed by 5 s.v. of 0.5x Kk 125 buffer (w/ EDTA), followed by 0.5x Kk metal 125 buffer (minus EDTA). Then I flowed in the 4.s.v of 1:484 EcoRI (5x NEB)

12:08AM, Flowed in 5 s.v. of 1x Kk Buffer supplemented to 50 mM EDTA. The hope here is to leach all of the metal ions out of the BGB and whatever else is in the sample. I'll let sit for a few minutes, and then re-try the 0 Ca 0 EDTA 0 BGB experiment

12:27AM, FLowed in 5 s.v. of 0.5x Kk metal buffer (0 mM EDTA)

12:29AM, another 5 s.v.

12:31AM, 4.4 s.v of EcoRI 1:484 in Kk metal 125 (0 Ca, 0 EDTA)

0145-0148	EcoRI 1:484 in Kk metal buffer 125 mM NaCl, 0 mM Ca, (0 mM EDTA is implied by "metal" label); 100 kHz / s, j600 F15

0149-0150	After the leaching attempt (50 mM EDTA). There are very few tethers left, so I didn't keep taking data.

020311.txt
3/11/02 1:19 AM

Sample was prepared with typical popping based protocol. Again, like recently, I scrubbed the slides with windex; sprayed windex off with building air; rinsed with barnstead water; sprayed water off with building air. pBR322 was diluted 1:20.

Following typical popping based tethering, I washed sample with 2 x 5s.v. of "1x Kk 125 Minimal", which is 10 mM Hepes, 125 mM NaCl, and nothing else. Then I added the 4.4 s.v. of EcoRI 1:484 in the same buffer.

In the first group of samples, it appeared to have lots of high force popping, as if I still had contamination.

At 11:55 flowed in EcoRI in 1x Kk 125 Min 920Ca (which is the same with 920 micromolar of Calcium Chloride)

Found a small tether farm at 12:04 AM

12:14AM flowed in EcoRI 1:484 in Kk 125 min 9200Ca (9.2 mM Ca)

0151-0153	EcoRI 1:484 in Kk 125 minimal buffer (Hepes and NaCl only); 100 kHz / s reference

0154-0156	EcoRI 1:484 in Kk 125 min 920Ca

0157-0160	EcoRI 1:484 in Kk 125 min 9200Ca

020312.txt
3/12/02 6:56 PM

Top sample is Bsa-popping buffer protocol (replacing BGB with NEB BSA 10 mg / ml)

Bottom sample is BGB-popping based.

Preliminary analyses: BSA sample has superior tether to stuck ratio, with about the same number of tethers. That is, it appears to have worked beautifully.

5:47 PM, Flowed into the BSA sample 4.4 s.v. of EcoRI 1:484 in Kk 125 min 0 Ca, after washing with 2 x 5s.v. of 1x Kk 125 minimal. AFter flowing in new buffer and EcoRI, the tether density seemed to go down DRASTICALLY. IN contrast, the BGB sample retained tethers later on.

[Note added a week later: I would guess that the decrease in tether density was a mistake. The repeat of this experiment showed a lower tether density to begin with.]

6:36PM, flowed 1:484 EcoRI in Kk 125 buffer (full compenents, except BGB) into the BGB sample. I did not pre-rinse with Kk buffer before flowing in the 4.4 s.v. of EcoRI

0161-0162	BSA sample; 1:484 EcoRI in Kk 125 min 0 CA

100 kHz reference

0163	same sample, switching to 1 MHz ref.

I think I am just seeing no binding.

0164-0166	1:22 EcoRI

0167-0169	BGB sample; Still did not see much popping, but tether density was reasonably high (indicating that RE cutting was not a problem, at least in this sample.

020315.txt
3/15/02 12:32 AM

Both samples on same glass, cleaned with windex scrub / h2o rinse method (building air). Both sampels I flowed through popping buffer before antidig.

Sample A "Bsa BLocking": Like normal sample, except BGB 5 mg / ml replaced with 10 mg / ml BSA from NEB.  The DNA is 1:20 pBR322 in NEB BSA

Sample B "BGB Blocking": Like normal, except pBR322 diluted in 5 mg / ml BGB.

Tethering efficiency:

BSA Sample, large (200 micron) field of view:

Stuck / tether: 1/2; 4/3; 6/1; 3/0; 5/1; 1/0; 0/1; 4/0; 0/2; 11/1

BGB Sample:

s / t : 5/ 7; 3/8; 2/10; 5 / 5; 5 / 2

11:19PM Flowed the EcoRI 1:484 into the BSA Sample

11:48PM Flowed in EcoRI 1:484 in Kk Metal 920micromolar Calcium

12:20 AM Flowed into BGB Sample EcoRV, 1:50 in 1x Kk buffer. THe EcoRV is Gibco Lot # 1069588, which is 10,000 U / ml stock

0170-0171	BSA Sample: EcoRI 1:484 in Kk buffer (no BGB or extra BGB supplementation).

Still shows no occupancy!!!

0172-0173	Same sample, 50 times faster stretching

0174-0175	EcoRI 1:484 in Kk metal 920 Ca.

Still very low occupancy, although I probably DID see some binding (and high force even)

EcoRV/0002	low stretch rate

EcoRV/0003	high stretch rate

020318.txt
3/18/02 11:45 PM

BGB Tethering protocol.

Flowed in EcoRI 1:484 in Kk 125 (no BGB) supplemented to 10 mM EDTA. However, I made a pipeting error such that I put in a total volume of 46 ul instead of 44 ul. So, the dilution is a little higher, and the salts etc. are a little off. I flowed in at about 10:43:30 PM.

I found a small tether farm right away (indicating that windex treatment was not 100%??? Anyway, it was also easier to flow the initial PBS into the sample, which would say glass wasn't "super clean")

11:17 PM, FLowed into the same sample 4.4 s.v. of 1:484 EcoRI in Kk 125, including 500 ug / ml BGB (so, the same buffer I used to take data for the paper).

The BGB sample definitely seemed to have very high occupancy at the beginning. However, it subsequently seemed to drop. Then I moved back towards the center, and it may or may not have increased again.

I WONDER: Are the narrower samples (e.g., slides with two chambers) more prone to non-specific sequestering of the EcoRI at the edges. For example, sticking to the double-stick tape edges?

NOTE: This slide was actually a dual sample chamber slide (and even particularly skinny), even though I only prepared the bottom sample.

0176-0179	EcoRI 1:484, 10 mM EDTA

0180-0181	EcoRI 1:484, 1 mM EDTA 500 ug / ml BGB

0182-0183	Same as previous two segments, except that I ensured that I am more near the center (most flow) of the sample.

020319.txt
3/19/02 6:20 PM

Double-wide sample (more than 20 microliters, probably). Normal tethering protocol with volumes doubled. (BGB blocking)

Flowed in 1:484 at 5:32PM in Kk 125 supplemented with 500 ug / ml final of BGB

In the center of the sample, occupancy seemed fairly high, although perhaps slightly low (but definitely much higher than 50%)

0184-0187	EcoRI In the middle of the sample

0188-0189	Near the top edge of the sample

0190-0191	Back in the center again (seemed like less occupancy...there is danger that I was "re-mowing" the same grass.

020322.txt
3/22/02 2:08 AM

This will be the first data set with Jen-Jacobson EcoRI. The source is the HCDB (a small dilution into 50% glycerol). It will be 1:6400 dilution.

The sample is normal popping / BGB based. I did the windex scrub / building compressed air cleaning method, and I must say that the sample at first (before Eco) looks very nice.

Flowed EcoRI in (1x Kk 150 buffer (no BGB or Ca) at 1:01:30AM

For whatever reason, clearly not enough protein. Will try higher concentration.

1:08AM, replaced with 1:80 EcoRI (note, will have a higher proportion of the storage buffer)

1:33AM, flowed in 1:80 in Kk 150 plus 0.02% tween-20

It does not look like the Tween helped at all.

0194	1:6400

0195-0196	1:80 in Kk 150

0197-0200	1:80 in Kk 150 plus 0.02% tween

020326.txt
3/26/02 2:24 PM

Popping based protocol, 1:20 of pBR322 in popping buffer.

Beads and antidig are from last week dilution.

Added 1:3600 JJ EcoRI in Kk150 (with BGB) at about 1:33:30PM.

At 2:04 PM, replaced the JJ EcoRI by flowing in 6 s.v. of 1:3600 NEB 5xEcoRI in Kk 150 (with BGB)

0201	FJ 10MHz/s; JJ 1:3600

0202	VC 10MHz/s; JJ 1:3600

0203-0204	Fadjust 10MHz/s; JJ 1:3600

0205-0208	Fadjust 10MHz / s; NEB 1:3600

020326_2.txt
3/26/02 5:33 PM

Trying to as closely resort to old protocol as possible. Changes from last sample:

Slides cleaned with water only, no scrubbing (I even used polypropylene gloves while cleaning). New double-stick tape as well (on water-only cleaned slide)

I did the biotin quenching step (about 30 seconds with popping buffer bioton (so it has a little DMSO).

I also note that the coverglass are a new batch.

The 500 ul tubes are also a new container (don't know about batch)

Flowed in NEB EcoRI 1:3600 in Kk150 (BGB) at 5:06:30PM

5:27PM, Flowed in the same kind of EcoRI as before, except prepared in sterile tubes. (And dilution was 1:60 x 1:60, instead of 0.5:30 x 1:60 as before)

OK: I could swear that I saw something swimming (like backteria, except maybe too small)? In any case, that is terrible. So I'll go re-make everything, or at least most things. It's probably the BGB that is offending.

0209-0210	1:3600 NEB EcoRI

0211	1:3600 of the new batch

020327.txt
3/27/02 6:50 AM

Top Sample: BGB BLocking (Fresh BGB see lab notebook today)

Bottom Sample: Casein Blocking (Fresh)

Both samples popping buffer tethering, 1:20 pBR322

90 micron octagons:

Top: Stuck / Tether: 5/3; 7/2; 7/2; 8/2; 7/1

Bottom: S/T: 6/2; 3/1; 3/5; 8/1; 5/0

Both samples look similar, with extra stuck beads in both samples (compared to before the fresh BGB). Tether density is perhaps a little higher also.

Notes about sample prep: Glass cleaned only by squirting water and spraying with building air (no scrubbing or windex). I did not quench with biotin.

5:23:30AM, FLowed EcoRI (NEB 5x) 1:3600 into the top (BGB) sample.

About 5:48AM, Flowed EcoRI (NEB 5x) 1:3600 into the bottom (Casein) sample.

6:31:30AM, Flowed in the rest of the 1:60 NEB EcoRI into the casein sample. There may be a large bubble preventing good flow, however. (BTW, by this time, most of the tethers seem to have gone. I would guess detached (or cut), as opposed to stuck, since the overall density seems lower of stuck + tether)

0212-0214	BGB Sample, EcoRI 1:3600; Fast

0215-0218	Casein Sample, EcoRI 1:3600; Fast

0219	Casein Sample, EcoRI 1:3600, DFS

0220-0221	Casein Sample, EcoRI 1:60 DFS

020330.txt
3/30/02 5:53 PM

Aptamer project

In the 240 pM 2:1 sample (see lab notebook), I see no obvious "tethers"...perhaps some jiggling,...

90 micron:

(Stuck or Jiggling) / (really jiggling): 5/0; 1/0; 3/0; 8/0; 2/0

10:1 sample: 1/0; 4/0; 7/0; 4/0; 3/0

After adding 10x more complexes to the 10:1 sample, I did not really see an appreciable increase in bead surface density. So, I conclude that these samples don't work at all

020330_2.txt
3/30/02 8:28 PM

8:13 PM, flowed in 10 s.v. of BsoBI 1:100 in Kk150 (+BGB)

File Popping_VC_BsoBI/today

The occoupancy is just too low with these particular conditions. I can either try Kk 100, or resort to the old popping buffer.

0033	0.1MHz/s reference

0034	0.5 MHz / s reference

020331.txt
3/31/02 4:54 PM

Typical Popping based tethering protocol, 1:20 of HindIII-capped internal biotin. I included the biotin quench step. I forgot to sonicate the beads, which I diluted freshly today.

4:29PM, flowed in 1:50 BsoBI in popping buffer

Wow. I forgot to sonicate the beads. Is that why there are no tethers? There are very, very, very few tethers (I found 3), and many of the stuck beads seem to be double beads or more. Hmmm.. I flowed in sonicated beads for about 10 minutes. WHile more stuck beads appeared (and single ones...not clumped), I didn't really see more tethers. Strange. I will just have to re-try the whole prep.

0035	0.5MHz / s reference; only 3 segments because tether density is so low

020331_2.txt
3/31/02 8:33 PM

6:34 PM flowed in 1:50 BsoBI popping buffer

Through 0040, Same conditions	500 ref

020331_3.txt
3/31/02 8:45 PM

8:32 PM, flowed in AvaI

through 0002	AvaI 500 ref

020401.txt
4/1/02 4:18 PM

3:32:30 added the AvaI 1:50 in pure popping buffer.

3:47 FLowed in AvaI 1:50 in popping buffer supplemented with 50 mM extra NaCl

4:06:30 AvaI 1:50 in Popping buffer plus 500 ug / ml BGB

Tether density is annoyingly low.

0003	forgot to turn on "steve params"

0004	10 segments, AvaI 1:50 in pure Pop Buf

0005	AvaI 1:50 in Pop plus 50 mM NaCl

0006	AvaI 1:50 in Pop plus 500 ug / ml BGB

020401_2.txt
4/1/02 6:03 PM

This is a new sample, prepared just like the previous, except replacing BGB with Casein.

The tethering density is still pretty low, but seems significantly better than the last sample. However, it's about as good as the best from yesterday (which was BGB based), so it's likely that the difference is random rather than casein versus BGB.

5:15:30 Flowed in AvaI 1:50 in Pop plus 500 ug / ml Casein

5:32:30 FLowed in Ava1 1:50 in Kk metal 920 uM Ca

5:46 Flowed in AvaI 1:10 in Pop plus 500 ug / ml Casein

0007-0008	AvaI 1:50 in Pop plus 500 ug / ml Casein

0009	AvaI 1:50 KkMetal 920 uM Ca

0010	Same as previous, 5x stretch rate (still seems to be low occupancy

0011	AvaI 1:10 in Pop plus casein, 500 Khz ref

0012	same as previous, 3x faster

020402.txt
4/2/02 2:01 PM

Typical popping based tethering, BGB replaced with casein. (HindIII capped 17-mer diluted 1:20 in popping buffer).

Tethering denisty is pretty low, but doable. Stuck beads also very low.

1:44 PM, Flowed in 1:50 BsoBI popping buffer

0040-0041	BsoBI 1.5 MHz/s reference

020402_2.txt
4/2/02 6:38 PM

Typical popping based tethering, BGB replaced with casein. (HindIII capped 17-mer diluted 1:20 in popping buffer). I flowed in 4 extra sample volumes of antidig, to see if that would increase the tether density. It seemed to have no effect. May want to try reducing the casein before the DNA

Tethering denisty seems lower than the previous sample today.

0042-0044	BsoBI 1500 reference

020409.txt
4/9/02 4:31 PM

Typical popping based tethering, BGB replaced with casein. (HindIII capped 17-mer diluted 1:20 in popping buffer). double the time on DNA and bead incubation steps (was talking)

Added AvaI (concentrated) 0.5ul in 25 ul of popping buffer at about 4:21PM

0017 and previous	naked DNA, screwing around with dF / dt clamp parameters

0018	AvaI, 1.5MHz/ s reference.

Even though using 5x concentrated, still seems to be very low occupancy.

020409_2.txt
4/9/02 5:01 PM

Same sample as the AvaI earlier

Flowed BsoBI in at about 4:41PM

0045	Data just for testing the dF / dt with max force

Hmmm...second segment did not seem to work with maxF

0048	re-trying at 60 pN. Problem is probably when the bead moves out of trap further, so that force is higher than we think, based on the setpoint.

0047	demonstrating that it works for maxF = 30 pN

0048	5MHz/ s

0049	3MHz / s

020508.txt
5/8/02 3:40 PM

Protocol can be found in data directory.

J-J EcoRI 1:50 in Kk200 added @ 3:05 PM

@ 3:25 PM, switched to J-J 1:50 in Kk150

0222	1:50 EcoRI in Kk200 dF / dt clamped

0223	1:50 EcoRI in Kk150 dF / dt clamped

0224	same sample, one data set VC clamping only

0225-0226	same sample, back to dF / dt clamp, 0.1 reference

020513.txt
5/13/02 4:42 PM

EcoRI this morning on 17-mer.

Protocol is all casein based, popping buffer, except for last step. I used casein in ALL steps at 5 mg / ml in popping buffer except EcoRI step, where I used 500 ug / ml in specified buffer. Even the DNA was diluted in 5 mg / ml casein (popping buffer, 1:20 17-mer HindIII capped)

J-J EcoRI 1:200 in Kk150 casein & tween; added @ 1:39 PM

0227-0229	EcoRI, 1 MHz reference

020514.txt
5/14/02 1:58 PM

J-J EcoRI on 17-mer.

Protocol is pretty much same as yesterday. Protocol is all casein based, popping buffer, except for last step. I used casein in ALL steps at 5 mg / ml in popping buffer except EcoRI step, where I used 500 ug / ml in specified buffer. Even the DNA was diluted in 5 mg / ml casein (popping buffer, 1:20 17-mer HindIII capped)

J-J EcoRI 1:400 in Kk150 casein & tween; added @ 1:13PM

By "Casein and Tween", I mean 500 ug / ml casein plus 0.02% tween

0230-0235	EcoRI, 0.3 MHz reference

0236	Some Restretching files, just to see how the 17-mer

behaves

0237	EcoRI, 0.3 MHz reference

020515.txt
5/15/02 5:28 PM

"Secret Peeking Software" was accidentally running during files 239 to 244 (or maybe 240-244)

J-J EcoRI on 17-mer.

Protocol is pretty much same as yesterday. Protocol is all casein based, popping buffer, except for last step. I used casein in ALL steps at 5 mg / ml in popping buffer except EcoRI step, where I used 500 ug / ml in specified buffer. Even the DNA was diluted in 5 mg / ml casein (popping buffer, 1:20 17-mer HindIII capped) The bead incubation step was over 1/2 hour long due to distractions.

J-J EcoRI 1:2000 in Kk150 casein & tween;

238-244	J-J EcoRI (1:2000) 0.3 MHz / s reference

020521.txt
5/21/02 6:12 PM

The first sample today, I forgot to store a "data set notes." The first sample was like the second one. They both had bubbles trouble. The first sample I forgot the casein wash after the beads, so I had to re-ad the eznyme later.

5:45 PM, second sample of the day.

Protocol is pretty much same as last week. Protocol is all casein based, popping buffer, except for last step. I used casein in ALL steps at 5 mg / ml in popping buffer except EcoRI step, where I used 500 ug / ml in specified buffer. Even the DNA was diluted in 5 mg / ml casein (popping buffer, 1:20 17-mer HindIII capped). Like before, the EcoRI is added in the wet lab, before mounting the sample. So, there is at least a 3 minute delay before the data set begins.

J-J EcoRI 1:2000 in Kk150 casein & tween

THis enzyme was diluted earlier today (the first of the two step 1:2000 dilution)

I don't know why, but this sample had a much lower density of stuck and tethers (sort of like the earlier one today). It may have been related to bubbles going through the sample. I should be more careful for that tomorrow. Maybe that also accounts for the high density of "crappy" tethers that I seemed to have stretched in this second sample today.

0245-0248	J-J EcoRI 1:2000 in Kk150 plus tween and casein

300 kHz reference; first sample

0249-0250	J-J EcoRI 1:2000 in Kk150 plus tween and casein

300 kHz reference; second sample

020525.txt
5/25/02 6:57 PM

Protocol is pretty much same as last week. Protocol is all casein based, popping buffer, except for last step. I used casein in ALL steps at 5 mg / ml in popping buffer except EcoRI step, where I used 500 ug / ml in specified buffer. Even the DNA was diluted in 5 mg / ml casein (popping buffer, 1:20 17-mer HindIII capped). Like before, the EcoRI is added in the wet lab, before mounting the sample. So, there is at least a 3 minute delay before the data set begins.

EcoRI added at about 6:18 or so

Overall, this sample very low tether density and crappier tethers. I incubated extra long on DNA and bead steps, don't know if that accounts for anything.

0251-0254	J-J EcoRI 1:2000; Kk150 plus casein and tween

020526.txt
5/26/02 1:20 PM

Protocol is pretty much same as last week. Protocol is all casein based, popping buffer, except for last step. I used casein in ALL steps at 5 mg / ml in popping buffer except EcoRI step, where I used 500 ug / ml in specified buffer. Even the DNA was diluted in 5 mg / ml casein (popping buffer, 1:20 17-mer HindIII capped). Like before, the EcoRI is added in the wet lab, before mounting the sample. So, there is at least a 3 minute delay before the data set begins.

EcoRI added at about 12:55 or so

Sample has a lot of "gunk" floating around, as if one of my solutions has gone bad. Gun has various forms, long skinny "sticks" (0.5 micron by 10's of microns) and spherical stuff (several micron diameter). None of it seems to be swimming.

0255-0258	J-J EcoRI 1:2000 Kk150 plus tween and casein

020526_2.txt
5/26/02 6:02 PM

Protocol is pretty much same as the All Casein protocol used for the past few weeks. However, I have replaced all Casein steps with freshly made BGB.

EcoRI added at about 5:35 or so

So, for this BGB sample, it did not seem to improve the number of "wacky" tethers. It seems that the majority of the tethers cannot go full length before going into a "weird" mode. One thing that I often see is the tether _almost_ break, followed by catching on something, and then a steady, jerky increase in force. I would imagine I have seen this at least 5% of the time, definitely more than that today, and I would expect it to show up in the data: force will be like unzipping / popping, then drop close to zero, followed by continuation of weirdness.

Since BGB and casein are likely similar, I can't really rule them out as the culprit. Although it does not seem to be the freshness that matters, since I just made fresh BGB. Perhaps I should compare also with NEB enzyme, and also with pBR322 construct?

One other thing to note, I suppose is that I could be using fairly old antidig, as I am almost running out, and only use 20 ul for each sample.

0259-0261	J-J EcoRI 1:2000 in Kk150 plus tween and BGB

020526_3.txt
5/26/02 8:45 PM

This is a dual sample, doing side by side comparison of pBR322 and 17-mer HIndIII capped. Both are with BGB and without EcoRI (last step is simply 5 mg / ml BGB popping buffer).



It is completely clear to me that these two samples behave very differently. The 17-mer is dominated by freaky tethers and crappiness. Conversely, the pBR322 sample has a large percentage (probably >50%) that stretch completely without any obvious weirdness. I did see a small 5-10% amount of weirdness in the pBR322 construct.

It is possible that it is simply a tether density effect. Since the 17-mer had slightly fewer tethers, perhaps I am biased towards finding weird stuff. Or it could be something intrinsic to the 17-mer construct.

In any case, it's probably not worth fooling around with the 17-mer under the conditions that I have been using.



8:31 PM, added J-J EcoRI 1:2000 (from older 1:44 dilution) to the pBR322 sample

0262-0263	17-mer; naked in 5 mg / ml BGB in popping buffer

0264-0265	pBR322; naked in 5 mg / ml BGB in popping buffer

0266-0268	J-J 1:2000 in Kk150 plus BGB plus tween; pBR322

020603.txt
6/3/02 6:19 PM

Popping Buffer / Casein protocol, similar to recent past.

5ul old capped 17-mer plus 5ul of popping buffer is what i'm tethering with.

BsoBI added at 5:35PM

With the 5x extra DNA, I do indeed get more "non-crappy" tethers. Probably the tether density was reduced somewhat by using old beads.

0050	1MHz / s

0051	2 MHz / s

0052-0053	5 MHz / s

0054	2 MHz / s

020621.txt
6/21/02 1:30 PM

Popping Buffer / BGB protocol, similar to recent past.

DNA is P1-dig-F and P13-bio-R PCR product (double tether)

Purpose is to do a DFS control, and show that other linkages are stronger than the DNA / protein interaction.

Oops, there are about 200 tethers per 90 micron octagon. So, I should use even a 1:50,000 dilution of my DNA (something weird is going on?).

0014	Just some practice data sets, I will need lower tether density to take the real control data.

020628.txt
6/28/02 1:02 PM

This sample sucked. Probably bad beads. I was trying to do the typical Popping / BGB 17-mer protocol. (2.5 ul of 17-mer in 7.5 ul of popping buffer). I will flow in fresh beads to see if I get better tethers.

So, I flowed in fresh 1:20 dilution (into 5 mg / ml BGB popping buffer) of bangs beads. I think, indeed, more tethers formed. So, I will remake the sample and take data (probably tomorrow).

0015	A naked DNA tether. The computer (Aatte) was behaving strangely. FTC was taking twice as long as normal, and then the system locked up when I tried to run Secret Peeking Software.

0016	more naked DNA after rebooting.

FIGURED OUT THE PROBLEM: My AOD Hard Limits frequency range had somehow gotten switched to 10 MHz. That was slowing down the FTC

020701.txt
7/1/02 10:04 AM

Typical Popping / BGB based. Diluting the HIndIII capped DNA 1:4 in 5 mg / ml BGB popping buffer. There is no biotin quench step. Stretching buffer is 1:100 BsoBI in straight popping buffer.

0018-0020	2 MHz / s df / dt

0021-0023	0.2 MHz / s dF / dt; Fresh BsoBI before hand

0024-0026	0.5 MHz / s dF / dt; Fresh BsoBI before hand

0027-0029	5 MHz / s dF / dt; Fresh BsoBI before hand

020701_2.txt
7/1/02 10:42 AM

Typical Popping / BGB based. Diluting the HIndIII capped DNA 1:4 in 5 mg / ml BGB popping buffer. There is no biotin quench step.

Previously, was the BsoBI sample today.

Stretching buffer is 1:100 AvaI (concentrated) in straight popping buffer.

0019-0020	Naked DNA (Same BsoBI sample, have washed with 5 s.v. Popping buffer (wait 10 minutes), then 5 s.v. again). This file shows that BsoBI is still hanging around somewhat.

0021	Added 1:100 AvaI (concentrated) at about 10:32 AM.

This file shows that AvaI does not bind strongly at this concentration (no detectable binding, really)

020708.txt
7/8/02 9:32 AM

0030	BsoBI 20 kHz / s reference. Deleted file, forgot to tape slide

0031-0033	50 kHz; fresh BsoBI added

0034-0036	2000 kHz; fresh BsoBI added before 0034

0037	10000 KHz / s ; fresh BsoBI added before 0037

020709.txt
7/9/02 12:53 PM

BsoBI 1:100 in Popping buffer

0038-0041	10 MHz / s

0042-0045	500 KHz / s; fresh BsoBI added before hand

0046-0048	100 kHz / s; fresh BsoBI added before hand

020906.txt
9/6/02 4:18 PM

XhoI 1:100 in popping buffer added before file 0049. The sample is sealed with nail polish

0049-0054	0.5MHz/s reference

0055-0057	0.05MHz/s reference

0058	5MHz / s reference

020909.txt
9/9/02 12:10 PM

XhoI 1:100 in popping buffer added before file 0059 at about 10:55 AM. The sample is sealed with nail polish

0059-0064	2 MHz / s

0065-0070	0.05 MHz/ s

New Directory: "Repetitive experimentation"

File 0071	same tether as file 0070, last segment in other directory

Testing how DFS would look on repetetive stretching

020910.txt
9/10/02 11:03 AM

XhoI 1:100 in popping buffer added before file 0059 at about 10:00 AM. The sample is sealed with nail polish.

(Incidentally, I looked at the sample from yesterday (24 hours old), and there were no viable tethers remaining. This means that the sample is indeed degrading fairly rapidly.)

Also as a note, all of these files lately (even including June and July) are with the HindIII-capped 17-mer. So, some of these data sets can actually be used for calibration as well.

0072-0073	200 KHz / s (Arthur and visitor were in the room talking)

0074-0077	200 KHz / s

0078-0082	5000 KHz / s

020911.txt
9/11/02 11:20 AM

BsoBI 1:100 added before file 0083. Sample is sealed with nail polish. (Note: this sample actually has a little less DNA than previous many weeks. I ran out of DNA today, so today used about 1.5 ul of DNA added to 7.5 ul of buffer (for 1.5 ul in 9 ul total).

0083-0088	200 KHz / s

0089-0091	50 KHz /s

021103.txt
11/3/02 2:34 PM

BsoBI 1:100 added before first file. BsoBI was in about 5-10 minutes before that. Sample is sealed with nail polish.

Protocol is normal Popping / BGB protocol. DNA is from July 2001, and is the "unpurified HindIII ligation attempt", diluted 1:2 today in 5 mg / ml BGB.

0092-0097	BsoBI loading rate clamp 500 reference. There is a strand of fuzz in the BFP, not sure which segments it may affect, if any.

0098-0100	5,000 reference

0101-0103	50 reference

021112.txt
11/12/02 12:43 PM

BsoBI 1:100 added before first file. BsoBI was in about 10 minutes before that. Sample is sealed with nail polish.

Protocol is normal Popping / BGB protocol. DNA is from July 2001, and is the "unpurified HindIII ligation attempt", diluted 1:2 today in 5 mg / ml BGB.

NOTE: The BGB is the same tube that is a couple weeks old. Also, I forgot to sonicate the beads today. For one or both of those reasons (or something else), the sample today is not nearly as nice as last Sunday. I see many more strange things, and the tethers seem to break earlier. Also, there are many more clumped beads stuck / tethers around. THis may be good to keep in mind, if the data end up looking strange.

104-109	500 kHz / s

110-112	10,000 kHz / s

021112_2.txt
11/12/02 4:03 PM

BsoBI 1:100 added before first file. BsoBI was in about 5-10 minutes before that. Sample is sealed with nail polish.

Protocol is normal Popping / BGB protocol. DNA is from July 2001, and is the "unpurified HindIII ligation attempt", diluted 1:2 today in 5 mg / ml BGB.

NOTE: The BGB is the same tube that is a couple weeks old. I remembered to sonicate the beads this time.

After taking all the data with the second sample, I wouldn't say the sample was any better than the first (except fewer clumped beads). So, perhaps it still is the BGB being old (as opposed to the lack of sonication)

113-115	5,000 KHz / s

116-118	200 KHz / s

119-121	100 KHz / s

122-124	2000 KHz / s

021120.txt
11/20/02 11:02 AM

XhoI 1:100 added before first file. XhoI was in about 5-10 minutes before that. Sample is sealed with nail polish.

Protocol is normal Popping / BGB protocol. DNA is from July 2001, and is the "unpurified HindIII ligation attempt", diluted 1:2 today in 5 mg / ml BGB.

The BGB was made yesterday with Dan (A difference is that I used room temperature popping buffer to make the BGB, whereas I usually use cold).

The antidig is several weeks old, I think.

0125-0131	2,000 kHz / s reference

0132-0137	5,000 kHz / s reference

0138-0142	50 kHz / s reference

0143-0145	500 kHz /s reference

030115.txt
1/15/03 4:36 PM

Protocol is normal Popping / BGB protocol. However, Popping buffer is made from DSJ 5x Popping buffer July 2002. So, THE pH IS NOW 7.5!!!

DNA is end-labeled from December 5 2001. I diluted 1:10 in BGB 5.

BGB made fresh today. Antidig diluted fresh today. Beads diluted fresh today.

Today will be Betaine studies. All buffers based on 1x Popping (DSJ), and all enzyme (if present) will be 1:100 dilutions of the BsoBI (same as from Papers)

The sample doesn't have very many tethers.

NEXT TIME use 1:2 dilution!!!

Will give up on this sample

0146	In naked Popping buffer

030121.txt
1/21/03 12:54 PM

Protocol is normal Popping / BGB protocol. However, Popping buffer is made from DSJ 5x Popping buffer July 2002. So, THE pH IS NOW 7.5!!!

DNA is end-labeled from December 5 2001. I diluted 1:2 in BGB 5.

BGB made fresh last week. Antidig diluted fresh last week. Beads diluted fresh today.

Today will be Betaine studies. All buffers based on 1x Popping (DSJ), and all enzyme (if present) will be 1:100 dilutions of the BsoBI (same as from Papers)

Sample has more tethers than with 1:5 dilution. However, the tethers don't seem to be full length (as if nicked)

0149? (Check time)	In 5 mg / ml BGB / popping buffer (DSJ)

0149-0150	Added BsoBI (5 s.v.) (1:50 in 1x DSJ Popping buffer) at about 11:46 AM, after washing with 5 s.v. of 1x DSJ Popping buffer

0151	Flowed in 5 s.v. more of BsoBI at 11:55 AM

0152	Re-trying with new buffer and enzyme. (Re-diluted 5x DSJ Popping buffer and re-diluted BsoBI in new 1x Pop) Flowed in 5 s.v. at about 12:10 PM

0153-0154	BsoBI 1:50 in SJK Popping buffer, flowed in 5 s.v. at about 12:19 PM

0155-0157	2/6 M Betain (1:6 dilution of 2M Betain in Popping) plus 1:50 BsoBI @ 12:30 PM

030124.txt
1/24/03 5:49 PM

Protocol is normal Popping / BGB protocol. However, Popping buffer is made from 2x Popping buffer January 2003. BGB is actually 10 mg / ml ish.

DNA is end-labeled from December 5 2001. I diluted 1:2 in BGB 10.

BGB made fresh today. Antidig diluted fresh last week. Beads diluted fresh today.

Today will be Betaine studies.

I will try probably only 1 M betaine 1x popping buffer plus 1:1000 BsoBI

16 amp laser, sum signal at 1.5 V AOD is 243 units.

0158-0159	1M betaine 500 kHz / s; 1:1000 BsoBI added at 4:19 PM

0160	repeating same tether

0161	repeating same tether with reversal

0162-0163	same as 0158-0159; added fresh enzyme before hand

0164	refolding last tether from 0163

0165	back to normal

0166	michelle data

0167-0168	michelle data, 10,000 reference

0169	same tether, michelle trying reversal at 10,000 initial

0170	same tether, michelle trying reversal at 500 initial

Untitled.txt
D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0025.dat	Contains example of 0.35 um peeler. Otherwise mostly junk

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0026.dat	Contains example of 0.53 um in BsaIBuffer

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0027.dat	Pre-BsaI, shows some examples of artifactual data

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0028.dat	Some Pre-BsaI, no good data

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0029.dat	Trying to gather Pre-BsaI data. Large File.

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0030.dat	BsaI--contains one good template

D:\Aatte\koch\data\Project 3 -- Overstretch and Y Templates\010326\0031.dat	BsaI--really contains no good templates