Fall 2005 Biosketch work

10/12/05
I redid the digest on Tuesday, still with the incorrect pairs of restriction enzymes. I was going to run the gel again today, after only one night of incubation. I realized my error after I had already set a new gel, so I threw out the digests, and wrapped up the unused gel in Saran wrap and refrigerated it. I did the digest a third time today, but I only had 10 micros of each miniprep left, and I accidentally added 3 instead of 0.3 micros of BSA to the R0052. I still added enough deionized water to have 30 micros total.

I am such a mess.

Tomorrow morning I plan to run the gel of these new digests.

In addition, yesterday, I transformed competent cells using R0052 minipreps and plated them onto amp and kan LB plates. I innoculated two colonies from the kan plate into tubes with LB and kanamycin, and tomorrow I will aliquot 1.6 mL from each into tubes, add 0.4 mL 50% glycerol to each, and freeze them in the -80. Yi-an did the same with *-transformed cultures.

10/13/05
The digest worked, surprisingly. I ran the gel for 49 minutes at 100V, and 20 more minutes at 120V, and I got the expected bands for reporter and promoter. I excised them and froze the gel slices for later.



Lane 1: 1 kb

Lane 3: 100 bp

Lane 5: Blue Heron mCherry digest

Lane 7: R0052 digest

In the evening, I performed the DNA extraction (using 300 micros of QB for each gel slice) and ligation (using 2 micros of extracted R0052 promoter DNA and 8 micros of extracted Blue Heron mCherry reporter DNA). I took 3 tubes of OneShot cells, let sit for 20 minutes, then inserted all 21 micros of ligation mix into one, 3 micros water into another (negative control), and 3 micros pUC19DNA into the third (positive control). I heat-shocked at 42 degrees for 30 sec, and then added 250 micros of SOC media to each. I left the tubes in the incubator-shaker for 1 hour, and then plated the ligation-transformed cells onto a LB kan plate and the water- and pUC19-transformed cells onto LB amp plates. I left the plates in the 37 degree incubator.

10/14/05
Today I got colonies on my ligation-transformed plate! I had planned to perform an EcoRI/PstI double digest and gel electrophoresis on miniprepped DNA from 10 randomly chosen colonies, in order to check for promoter plasmids which were single-digested and self-ligated. Austin suggested that I perform a colony PCR instead with 10 randomly chosen colonies. After I set up the PCR, Austin noticed that the colonies are on my plate were red, which already means successful assembly. Sweet.

Today, I also diluted stock IPTG into tubes at 0.5 M: 1.2 g in 10 mL water, aliquotted 1 mL per tube.



10/16/05
Ran a gel of PCRs.



Lane 1: 100 bp

Lane 2: 1 kb

Lane 3-12: Colony PCRs 1-10

Lane 13: (-)

It's weird that 4 of the lanes have no bands at all, but they all come from red-positive colonies. Plan to do an innoculation and miniprep before PCR and gel to retry.

Innoculated colonies 1-10 to be frozen with glycerol and as pellets tomorrow, but realized later that I forgot to add kanamycin to the innoculations.

10/20/05
Did innoculations again of 10 streaked colonies which were ligation-transformed. Will freeze in glycerol, and do miniprep for sequencing. Innoculated Plambda(R0051,amp), QPI265(JO6801,kan), QPI434(QO4520,kan), and mCb(JO6702,amp).

10/21/05
Miniprepped DNA from 10 innoculations of ligation-transformed colonies (#2,5,6,10 looked pink in liquid culture!) and from innoculations of 4 constructs. Set up double digests and PCRs. R0051 and QO4520 digested with SpeI and PstI, JO6801 and JO6702 digested with XbaI and PstI.

10/22/05
Ran gel of digests.



Lane 1: 100 bp

Lane 3: 1 kb

Lane 5: QO4520

Lane 7: R0051

Lane 9: JO6801

Lane 11: JO6702

I've got bands for JO6801 and JO6702, but nothing for QO4520 and R0051. I excised and purified the JO6801 and JO6702 "lower" bands.

I don't think the YK PCR program worked. When I came in, it said " Init denat -> 0m00, sample 119C, ramp 94.0C @ max." I took the tubes to another PCR machine on which I've already created an "insert check" program.

I ran an e-gel of the miniprepped DNA from those 10 transformed colonies.



Lanes 1-10: Colonies 1-10

Lane 11: 100 bp

Lane 12: 1 kb

It looks like I've got nothing!

Then I ran a manual gel of the PCR'd DNA. Accidentally added 5 micros of loading dye, but remained consistent.



Lane 1: 100 bp

Lane 2: 1 kb

Lane 3: (-)

Lanes 4-13: Colonies 1-10 (lost a little of colony 8 in loading lane 11)

The huge tear in the gel is a result of me dropping the gel in the hallway.

I prepared sequencing reactions with DNA from colonies 1 and 3 (R0052-Blue Heron mCherry ligation-transformation). I diluted primers 4 micros into 36 micros water. The tube strip is organized thus.

Tube 1: 1 + VF2 (PT001)

Tube 2: 1 + VR (PT002) Tube 3: 3 + VF2 (PT003)

Tube 4: 3 + VR (PT004)

10/23/05
Ran gel of digests.



Lane 1: 100 bp

Lane 3: 1 kb

Lane 5: R0051

Lane 7: QO4520

It sucks.

I performed a ligation transformation using my JO6801 and gel-purified R0051 from Yi-an, and plated them out with amp selection.

I re-innoculated cultures from R0051, QO4520, JO6801, JO6702 stocks.

10/24/05
I came in to find my innoculations sitting on the bench: I'd forgotten to leave them in the incubator. Sigh, a day wasted. My ligation-transformation yielded zero colonies, at least that I can see, while the negative and positive plates yielded the expected zero and a shitload. I'm thinking that I didn't have enough JO6801, given the faint band in my digest gel 10/22/05. I left the plates in incubation for another night.

Retried the ligation-transformation with 2 micros R0051 from Yi-an and 8 micros JO6801 purified on 10/22/05.

Miniprepped DNA from extra pellets I froze of R0051, QO4520, JO6801, JO6702 on 10/21/05.

Setup double digests with DNA minipreps of R0051, QO4520, JO6801, JO6702 from 10/21/05, with DNA minipreps performed today from the extra pellets of R0051, QO4520, JO6801, JO6702, and also with JO6801 DNA miniprepped from 10/08/05.

10/25/05
The ligation-transformation plate from 10/23 which I left in for a 2nd night had a lot colonies; however, so did the negative plate (transformed with water). This means they got contaminated when I took off the lids to look inside, even though I was under a burner. The ligation-transformation plate from 10/24 had maybe five colonies, the positive plate was covered, and the negative plate unfortunately looks like it has several colonies as well. I streaked 5 colonies from the lig-trans plate onto a new amp plate, and innoculated the toothpick with which I streaked.

I ran a gel of my digests from last night.



Lane 1: 100 bp

Lane 2: 1 kb plus

Lane 3: R0051 from 10/21 (only 10 micros miniprep)

Lane 4: QO4520 from 10/21 (only 6 micros miniprep)

Lane 5: JO6801 from 10/21

Lane 6: JO6702 from 10/21

Lane 7: R0051 from 10/24

Lane 8: QO4520 from 10/24

Lane 9: JO6801 from 10/24

Lane 10: JO6702 from 10/24

Lane 11: JO6801 from 10/08

I excised the lower bands from Lanes 5, 6, 9, 10, 11. I also saw and excised a very faint band in Lane 7. I purified and froze the DNA.

I miniprepped DNA from the new innoculations I incubated last night of R0051, QO4520, JO6801, and JO6702.

10/27/05
I miniprepped DNA from the innoculations of colonies from my R0051-JO6801 ligation-transformation plate. I PCR'ed this DNA with my "insert check" program.

10/28/05
I didn't have time yesterday to get out my PCR'ed DNA, but someone kindly took the tubes out out of PCR machine #1 and put them in the SynthBio freezer. However, when I went to add loading dye, I really don't think anything was in the tubes. And when I went to load the wells, I could only get out as much dye I'd put in (3 micros). Where'd my PCR reaction go?! I ran the gel anyway, in hopes something might show up.



Lane 1: 1 kb

Lane 3: l kb plus

Lane 5: (-)

Lane 7: R0051-JO6801 lig-trans colony 1

Lane 9: R0051-JO6801 lig-trans colony 2

There's nothing there. I setup the PCR reaction again.

I also ran an e-gel of miniprepped DNA of R0051 and QO4520 to check if there was any DNA at all. I usesd 3 micros of ladder, and 10 micros of miniprep + 3 micros dye.



Lane 1: 1 kb

Lane 2: 1 kb plus

Lane 3: R0051 miniprepped 10/24

Lane 4: QO4520 miniprepped 10/24

Lane 5: R0051 miniprepped 10/25

Lane 6: QO4520 miniprepped 10/25

It looks like R0051 and QO4520 from 10/25 have a significant amount of DNA. I setup double digests of both of those minipreps. This time I used a PCR machine #4, with this program: one loop at 37 degrees for 2 hours, with a hot start at 37 degrees, a (pre)heated lid at 105 degrees, only 1 sec of final extension at 37 degrees, and final hold at 4 degrees.

10/30/05
Ran a gel of digests and PCRs.



Lane 1: 1 kb

Lane 3: 1 kb plus

Lane 5: PCR (-)

Lane 7: PCR Colony 1 R0051-JO6801

Lane 9: PCR Colony 2 R0051-JO6801

Lane 11: R0051 SpeI/PstI digest

Lane 13: QO4520 SpeI/PstI digest

Excised R0051. Weird smears with QO4520 digest and with PCR Colony 2. PCR Colony 1 band is a little above the 100 bp band on the 1 kb+ ladder.

11/6/05
It's been a long time. I haven't been in lab all week b/c I've been really busy with my acappella group (Harvard LowKeys) rehearsing and preparing for our big KeyLime concert last night. Now back to the bench...

I prepared double digests using Katherine's 50 micro protocol. I used the midipreps of R0051 and QO4520 found in the "Synthetic Bio minipreps" box, and different minipreps I've made before of JO6702 and JO6801. I could only get 25 micros of the 10/24 JO6702 and JO6801 minipreps, and 15 micros of the 10/8 JO6801 miniprep. I added the appropriate amounts of additional water. I incubated the digests in a PCR machine for 2 hours at 37 degrees.

I prepared PCRs of miniprepped DNA from the colonies of R0051-JO6801 (hopefully) transformed colonies. I incubated the PCR reactions using Ezra's PCR program.

I ran a gel of the digests and PCRs.



Lane 1: 1kb plus

Lane 2: PCR (-)

Lane 3: PCR DNA from colony 1

Lane 4: PCR DNA from colony 2

Lane 5: R0051 midi

Lane 6: QO4520 midi

Lane 7: JO6702 miniprepped 10/24

Lane 8: JO6801 miniprepped 10/24

Lane 9: JO6702 miniprepped 10/25

Lane 10: JO6801 miniprepped 10/25

Lane 11: JO6801 miniprepped 10/8

The appearance of a band in Lane 2, PCR (-) at 300 bp is very odd. This may mean that something in my PCR master mix (PCR SuperMix + VF2/VRprimers) was contaminated. Specifically, I made a master mix, which I aliquotted into two tubes, to which I added DNA from Colonies 1 and 2. What was left of the master mix served as the negative control. This suggests that the negative control got contaminated after I aliquotted the master mix into tubes because the band does not appear in lanes 3 or 4. The band for colony 1 is a little more than 100 bp, too small to be a successful ligation-transformation. The band for colony 2 is a little under 1,650 bp.

I excised and purified gel fragments from lanes 5-11. Even though there was a smear in lane 5, the bright band at the bottom corresponds to the same size I've seen before for SpeI/PstI digested R0051, so I excised it anyway. I excised the only band in lane 6 for QO4520. I excised the lower bands for the other lanes of JO6702 and JO6801.

11/7/05
I performed two ligations.


 * R0051-JO6801 (Plambda-QPI265)
 * QO4520-JO6702 (QPI434-mCb[-LVA])

I used R0051 and QO4520 from "Synthetic Biology minipreps" which I digested and gel purified yesterday, 2 micros of each. I used JO6702 and JO6801 miniprepped 10/25, digested and gel purified yesterday, 8 micros of each.

I transformed OneShot cells and plated (+) pUC19 DNA transformed cells, (-) non-transformed cells, and the R0051-JO6801 ligation transformed cells onto amp plates (amp(1) 8/16), and QO4520-JO6702 ligation transformed cells onto a kan plate (11/5). I left the plates in the incubator at 1:00 am.

11/8/05
I got a good number of R0051-JO6801 and QO4520-JO6702 ligation transformed colonies on my plates (checked around 4:00 pm). I used sterile toothpicks to streak out 10 colonies each and dropped the toothpicks into liquid LB for innoculation (+amp for R-J and +kan/IPTG for Q-J assemblies).

11/9/05
I spun down two pellets from each innoculation, one for freezing and one for miniprep. I prepared PCR reactions with the minipreps, including three negative controls, each from a different "PCR SuperMix" tube to check for the contamination found in the last PCR. My master mix for the rest of the PCR reactions came from PCR SuperMix tube #2. I used Ezra's PCR program on PCR machine #3.

I ran a gel of the PCRs.



Lane 1: 1 kb plus

Lane 2: (-) PCR SuperMix tube #1

Lane 3: skipped (noticed a disfiguration or break in the well)

Lane 4: (-) PCR SuperMix Tube #2

Lane 5: (-) PCR SuperMix Tube #3

Lanes 6-15: R0051-JO6801 transformed colonies 1-10



Lane 1: 1 kb plus

Lanes 2-11: QO4520-JO6702 transformed colonies 1-10

11/13/05
Because of the possible primer contamination, and because of the strange appearance of other bands in the R0051-JO6801 PCR gel and the odd sizes of the bands in the QO4520-JO6702 PCR gel, I reperformed the PCR with new VF2/VR primers on R0051-JO6801 colonies #2, 3, and 9, and on QO4520-JO6702 colonies #2,5, and 6. I also added two more negative controls using newVF2/oldVR and newVR/oldVF2.



Lane 1: 1 kb plus

Lane 2: (-) PCR, new VF2/new VR

Lane 3: (-) PCR, new VF2/old VR

Lane 4: (-) PCR, new VR/old VF2

Lane 5: R0051-JO6801 colony 2

Lane 6: R0051-JO6801 colony 3

Lane 7: R0051-JO6801 colony 9

Lane 8: QO4520-JO6702 colony 2

Lane 9: QO4520-JO6702 colony 5

Lane 10: QO4520-JO6702 colony 6

11/15/05
I performed a 50 micro double digest of the R0051-JO6801 assembly using SpeI and PstI. (used 25 micros DNA, added 5 more micros water)

I prepared sequencing reactions for R0051-JO6801 and QO4520-JO6702 assemblies.

Tube 1: R0051-JO6801 colony 2, VF2

Tube 2: R0051-JO6801 colony 2, VR *

Tube 3: R0051-JO6801 colony 3, VF2

Tube 4: R0051-JO6801 colony 3, VR *

Tube 5: QO4520-JO6702 colony 2, VF2

Tube 6: QO4520-JO6702 colony 2, VR

Tube 7: QO4520-JO6702 colony 5, VF2

Tube 8: QO4520-JO6702 colony 5, VR


 * For tubes 2 and 4, I could only get 3 micros out, since I'd just used 25 micros for the digest. I miniprepped more DNA from leftover pellet 11/9, and added 5 more micros from that.

I ran a gel of the digest.



The bands are faint but present between 3 and 4 kb. I excised those bands and purified the DNA (accidentally used 300 micros of isopropanol, but shouldn't affect the yield, according to the manual)

I realized right before the last step of gel extraction/purification (after adding 0.75 mL PE) that this whole day my centrifuge machine was on "g" mode instead of "RPM" mode. I should have realized it earlier when my miniprep lysed pellet seemed smaller than usual. This means my miniprep and most of my purification were run at 14,000 g instead of 14,000 RPM (while 17,900 g = 13,000 RPM).

11/16/05
Nanodropped some of the minipreps from the innoculations of 10 R0051-JO6801 and 10 QO4520-JO6702 colonies, and got very low numbers, Ran digest of QO4520 midiprep from "Synthetic Bio minipreps" box using XbaI/SpeI to check part size.

11/17/05
Prepared innoculations of 10 R0051-JO6801 and 10 QO4520-JO6702 colonies from the plated stocks. Streaked carb plate of R0052-BHmCherry colonies #2, 5, 6 from plated stocks and from glycerol stocks. Realized as I'm typing this that the colonies should be kan and not amp resistant. Streaked a kan plate of the same.

Ran gel of QO4520 midiprep XbaI/SpeI digest.



Lane 1: 1 kb plus

Lane 3: QO4520 XbaI/SpeI digest

Bands look correct for plasmid 4425 bp and part 903 bp.

Reperformed ligation/transformation with QO4520 purified 11/6 (DNA from midiprep) and JO6702 purified 11/6, miniprep 10/25.

Diluted more IPTG into tubes at 0.5 M: 1.2 g in 10 mL water, aliquotted 1 mL per tube.

11/18/05
R0052-BHmCherry colonies streaked from from plated stocks 2 and 6 are red, but none of the colonies streaked from plate stock 5 and from glycerol stocks 2, 5, and 6 are red. Centrifuged down the innoculations to pellets. Took transformation plate out of incubator and refrigerated.

11/20/05
Miniprepped DNA from pellets. Nanodropped several of them, all of which had >2.00 for 260/280 and <10 ng/ul. Performed PCR. Only 20 tubes fit in PCR machine, so left out R0051-JO6801 colony 6 PCR to fit in the negative tube. Innoculated cultures from colonies on Q04520-JO6702 transformation plate from 11/17/05. Using a Microcon centrifuge filter, I tried concentrating the miniprep from R0051-JO6801 colony 9. I added water to what I think was the collected concentrate (although I couldn't see anything in the vial), and then I centrifuged it after vortexing without labelling and stupidly used the flowthrough vial as a balance, and so I didn't know which was which. One had 15 micros in it; the other had 10 micros. I thought I'd added 15 micros water, but I wasn't sure anymore. Anyway, I set up a SpeI/PstI double digest, with the mix of 30 micros total volume, with each of them.

11/21/05
Ran gel of PCRs.



Lane 1: 1 kb plus

Lanes 2: PCR (-)

Lanes 3-7: R0051-JO6801 PCRs 1-5

Lane 8: skipped for R0051-JO6801 PCR 6

Lanes 9-12: R0051-JO6801 PCRs 7-10



Lane 1: 1 kb plus

Lanes 2-11: QO4520-JO6702 PCRs 1-10

Lane 12: QO4520-JO6702 digest from Microcon concentrated (10 ul)

Lane 13: QO4520-JO6702 digest from Microcon concentrated (15 ul)

Centrifuged two pellets from each innoculation of QO4520-JO6702 transformed 11/17/05. Miniprepped one of the pellets using new miniprep kit.

Cut out band from lane 9 in second gel (QO4520-JO6702 PCR 8). Plan to purify and sequence.

11/23/05
Prepared innoculations of QO4520, JO6702 from iGem stock and from Yi-an's stock, and R0051-JO6801 colonies 1-3, 5, 8-10. Used 3 micros kanamycin for 3 mL LB in QO4520 culture.

11/24/05
Pelleted innoculations twice in one tube: filled tube 1.5 mL culture, centrifuged, poured off supernatant, filled again with 1.5 mL culture, centrifuged, poured off supernatant. QO4520 culture did not grow very well Too much kanamycin? Tube of JO6702 iGEM culture "hopped" out of tube rack and spilled half of first step.

Miniprepped the DNA.

11/27/05
Prepared 50 micro digests, incubated for 2 hours, added phosphotase, incubated for one more hour, and ran gel.



Lane 1: 1 kb plus

Lane 2-8: R0051-JO6801 S/X digests, from 11/24 minipreps from colonies 1-3,5,8-10



Lane 1: 1 kb plus

Lane 2: QO4520 S/X digest, from 10/24 mini (15 micros DNA)

Lane 3: QO4520, class midi

Lane 4: QO4520, from 10/25 (15 micros DNA)

Lane 5: QO4520, from 11/24

Lane 6: JO6702, from 10/8 (did not load well into gel)

Lane 7: JO6702, from class midi

Lane 8: JO6702, from 10/21 (10 micros DNA)

Lane 9: JO6702, from 10/25 (5 micros DNA)

Lane 10: JO6702, from 11/24 mini (iGEM)

Lane 11: JO6702, from 11/24 mini (Yi-an)

I cut and purified DNA from QO4520 from class midi, QO4520 from 10/25, and JO6702 class midi. I performed three ligation/transformations.


 * QO4520 from class midi, purified 11/27 with JO6702 from class midi, purified 11/27
 * QO4520 from class midi, purified 1/27 with Blue Heron mCherry, purified 10/13
 * R0051 from class midi, purified 11/6 with JO6802 from 10/25 mini, purified 11/6

11/28/05
Q-B plate had one colony. Q-J plate had three colonies. R-J plate is covered with hundreds of colonies. I streaked out new colonies and prepared innoculations from the Q-B and Q-J colonies and 10 of the R-J colonies.

11/29/05
Miniprepped innoculations (R0051-JO6801 colonies 9's and 10's DNA may have gotten mixed up in labelling). Setup PCR.

11/30/05
Ran gel of PCR reactions.



Lane 1: 1 kb plus

Lane 2: (-)

Lanes 3-12: R0051-JO6801 colonies 1-10

Lanes 13-15: QO4520-BHmC colonies 1-3

Lane 16: QO4520-JO6702 colony 1

12/2/05
Looked at sequencing results with Chris. R0051-J06801 sequences look correct. Q04520-J06702 do not, as expected. Chris revealed to me that I've been working on incompatible halves of the assembly (R0051 and Q04520 are Plambda and QPI434). So because I have a successful R0051, I need to switch from Q04520 to Q04510, which is QPIlambda.

I took all the minipreps of R0051-J06801 from 11/29, pooled them into one tube, and concentrated the tube using the Microcon device.

I prepared digests of the R0051-J06801 pooled DNA, Q04510 class midiprep, and J06703 class midiprep. Incubated for 2 hours, asked Yi-an to add phosphotase to R-J and Q digests and incubate for another 2 hours.

12/3/05
Ran gel of digests.



Lane 1: 1 kb plus

Lane 3: Q04510 digest

Lane 5: J06703 digest

Lane 7: R0051-J06801 digest.

Purified Q04510 and J06703.

Prepared two 5mL innoculations of R0051-J06801 from colony 1 streak. Added IPTG to each.

Prepared two 3mL innoculations of R0052-J06704, one from colony 2 streak 10/14, and one from a random colony on initial transformation plate 10/13.

Performed three ligation/transformations: Q04510 from today with JO6702 from 11/27, J06703 from today, and JO6704 (BHmC) from 10/13. After I added SOC media and began initial growth, I couldn't find any kan plates! After the initial growth finished, I left the tubes in a refrigerator, with hopes to try to plate them out later. We'll see.

I prepared another digest of Q04510 from Yi-an's midiprep since I used it all in the triple ligation. I used 42.5 micros of DNA and no water.

12/4/05
This morning, R0052-JO6704 innoculations don't grow very well, and for good reason: I added ampicillin instead of kanamycin to the innoculation. I prepared two more innoculations with kanamycin and IPTG.

Pelleted both innoculations of R0051-JO6801 into one tube and miniprepped. Still yielded a low 22.8 ng/ul. WHYYYYYYYYY

Ran gel of Q04510 digest.



Lane 1: 1 kb plus

Lane 3: Q04510 digest

Cut and purified the DNA.

Prepared 200 mL innoculation of R0051-J06801 for a midiprep.


 * Hey Perry, the only thing I can think about for why your miniprep may be yielding low DNA is the growth conditions of your cells. You can Nanodrop your cultures to get the OD reading before your next miniprep and see how well your cells are growing. If you grow your cells too long, your cells will start dying and the prep will be bad. Conversely, if the cells aren't growing too well, you won't get much DNA. Another suggestion I have is to try moving or assembling parts into the plasmids I brought (e.g. pSB1AC3-1). If for example, there has been a mutation in one of the part's plasmid that determines its copy number, then this mutation would pass through during assembly. If you use the fresh plasmid, you won't have this issue. If you just want to move a part, you can cut both the plasmid and the part with EcoRI/PstI and do the ligation like that. --Austin 18:30, 4 December 2005 (EST)

12/5/05
Discarded midiprep b/c left in incubation for over 30 hours.

Split up innoculations (also over 30 hours, but still looked red; culture from random colony looked more red in liquid culture than culture from streak 2) from random colony and from streak 2 into 1 mL aliquots and pelleted.. Resuspended pellets in 200 micros PBS.

C1-3: random, C4: PBS, D1-3: streak 2

Made fluoresence measurements.

12/6/05
Made another 200 mL innoculation of R0051-JO6801 for midiprep and two more innoculations of R0052-JO6704 for fluorescence measurements later tonight.

I am the stupidest person alive. I again realized that I innoculated R0052-JO6704 with amp and not kan. GAH.

Midiprepped R0051-JO6801. Split up innoculation into 5 tubes, 1-4 of 35 mL and 5 of about 30 mL. After pelleting, added 2 mL P1 to each tube, pipetted suspension 2 into 1, 4 into 3, and split up 5 between 1 and 3. Applied supernatants from each to QIAGEN-tip, and then proceeded as printed. Dissolved DNA in 1 mL 10mM Tris-HCl.

I nanodropped the midiprep, and got 29.6 ng/ul.

I think that I will go throw myself out the window.

I prepared a phosphatase double digest using 42.5 micros of the midiprep.

I prepared 3 mL R0052-JO6704 innoculations from random colony/streak2, this time with kanamycin and IPTG. I prepared a 200mL innoculation of R0051-JO6801 again for another midiprep, after I plan to speak with Yi-an or Kristi.

Prepared graphs of fluorescence data from yesterday.

12/7/05
I was told that I dissolved my DNA in too large of a volume, so I used Microcon to concentrate my midiprep down to 100 micros water. Nanodropped, and got 202 ng/ul!!! Someone get the camera, it's a three digit number! I prepared another phosphatase double digest using 42.5 micros of this concentrated midiprep.

I streaked another plate from ten R0052-J06704 colonies because my stock plate is rather old.

I performed the same triple ligation-transformation as I had tried on 12/3: Q04510 from 12/3 with JO6702 from 11/27, J06703 from 12/3, and JO6704 (BHmC) from 10/13. However, I used the wrong proportion of vector and insert: 8 micros vector, 2 micros insert. So I did the triple ligation-transformation again with 2 micros vector, 8 micros insert. Wow, I'm wasting so many OneShot tubes.

I ran a gel of the digests of the original R0051-J06801 midiprep and the concentrated midiprep.



Lane 1: 1 kb plus

Lane 3: R0051-J06801 digest from original midiprep

Lane 5: R0051-JO6801 digest from concentrated midiprep

I extracted DNA from the concentrated midiprep band. Got around 40 ng/ul.

Meanwhile, I did another midiprep of R0051-J06801. This one turned out even worse: 11.6 ng/ul, and I dissolved in 0.5mL instead of 1mL this time. I am so angry. What could have been made it worse? I did do a few things different. I added IPTG several hours before I started the midiprep. I used the polycarbonate centrifuge tubes for the initial steps. I dissolved the DNA with TE instead of Tris-HCl.

12/9/05
I came in to find my tube of digested/extracted R0051-J06801 sitting on the bench: I'd forgotten to put it in the freezer!!! Gah, I hope it's not ruined; but I can do another digest just in case.

I got a bunch of colonies on my Q04510-mCherry transformation plates, and interestingly, I got more colonies on the transformation from ligation #1 with 8 micros vector and 2 micros insert than from ligation #2 with the reverse. However, none of the colonies are red. I streaked and inoculated from 3 colonies on each plate (3 different transformations from two separate ligations, so 6 plates, and 18 streaks/inoculations).

Also inoculated R0052 from a stock plate in 12 mL culture.

12/10/05
Miniprepped Q04510-mC and prepared PCRs. Miiprepped R0052 inoculation and prepared double digest, as well as double digests of concentrated R0051-J06801 midiprep, and of J06803 from 10/8.

Ran gel of PCRs.



Lane 1: 1 kb plus

Lane 2: (-)

Lanes 3-5: Q04510-J06702 ligation #1, colonies 1-3

Lanes 6-8: Q04510-J06702 ligation #2, colonies 1-3

Lanes 9-11: Q04510-J06703 ligation #1, colonies 1-3

Lanes 12-14: Q04510-J06703 ligation #2, colonies 1-3

Lanes 15-17: Q04510-J06704 ligation #1, colonies 1-3

Lanes 18-20: Q04510-J06704 ligation #2, colonies 1-3

Ran gel of digests.



Lane 1: 1 kb plus

Lane 3: R0052

Lane 5: J06803

Lane 7: R0051-J06801

Cut out bands of R0052 and R0051-J06801.

Did some fluorescence measurements with R0052-J06704. inoculated four nights ago (yes, four nights. I just sort of left them in there b/c I didn't have time to do anything with them, but hey, they were still red). got bizarre results.

fixed ex 570/max em 570 and fixed em 610/max ex 610.

fixed ex 610/max em 610, and fixed em 570/max ex 570.

then when i took endpoint measurements at 570/610 and 610/570, i got values in the hundreds, and when i took endpoint measurements at 570/570 and 610/610, i got values in the hundred thousands.

I prepared new PCR reactions of the Q04510-mCb minipreps that did not yield bands, this time with 2 micros of miniprep, and longer elongation period.

12/11/05
I prepared completely new 12 mL inoculations of Q04510, J06803, J06702, and J06703. Also prepared a 9 mL inoculation of random R0052-J06704 colony for a 45/25/4 degree fluorescence experiment.

12/12/05
Miniprepped Q04510, J06803, J06702, J06703. Prepared double digests.

Ran gel of PCRs from yesterday.



Lane 1: 1 kb plus

Lane 2: (-)

Lanes 3-5: Q04510-J06702 ligation 1, colonies 1-3

Lane 6-7: Q04510-J06702 ligation 2, colony 1-2

Lane 8: Q04510-J06703 ligation 2, colony 3

Lane 9: Q04510-J06702 ligation 2, colony 3

Lane 10-11: Q04510-J06703 ligation 1, colonies 1 and 3

Lane 12-13: Q04510-J06703 ligation 2, colonies 1-2

Lane 14-15: Q04510-J06704 ligation 2, colonies 2-3

Ran gel of digests.



Lane 1: 1 kb plus

Lane 3: Q04510

Lane 5: J06702

Lane 7: J06703

Lane 9: J06803

I cut out the Q04510 band (before I took the picture, obviously).

I purified Q04510 band from today, and the R0052 and R0051-J06801 bands from 12/10.

I ligated/transformed with Q04510 purified from today and JO6702 from 11/27, J06703 from 12/3.

I prepared digests with class midipreps of J06702, J06703, J06803.



Lane 1: 1 kb plus

Lane 3: J06702

Lane 5: J06703

Lane 7: J06803

Cut and purified all three bands.

12/13/05
I placed my transformation plates upside down (agar side down) last night, so I didn't get good colonies.

I performed three ligation transformations.
 * Q04510 12/12, J06702 12/13
 * Q04510 12/12, J06703 12/13
 * R0052 12/12, J06803 12/13

I prepared two 6mL inoculations from the new R0052 plate that Austin brought.

I tried to get a fluorescence spectrum with Austin's advice of keeping the fixed ex/em a distance from the sweeping em/ex.

12/14/05
Miniprepped new R0052 inoculations. Prepared phosphotase digest.

Ran gel of R0052, along with a few of Yi-an's digests.



Lane 1: 1 kb plus

Lane 3: R0052 (S/P phosphatase digest, 4463)

Lane 5: J06702 (YK, X/P digest, 2079 and 869)

Lane 7: J06800 (YK, X/P digest, 4425 and 1371)

Lane 9: Q04510 (YK, S/P digest, 5404)

Lane 11: R0051 (YK, S/P digest, 2120)

Cut out and purified DNA from top R0052 band and bottom J06702 band.

Performed ligation/transformation with R0052 from today and J06803 from 12/13.

Streaked and inoculated colonies from yesterday's transformation plates: 1 from R0052-J06803, 4 from Q04510-J06702, 4 from Q04510-J06703. Still no red colonies.

12/15/05
No colonies from new R0052-J06803 transformation. Miniprepped inoculations. I think I got some of the Q04510-J06703 colony 2 culture into the colony 3 culture during pelleting by mistaken pipetting. I prepared PCR reactions. Pellets of Q04510-J06702 looked a little pink? May have been my imagination.