User:GeorgeXu

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[edit] Important Stuff that was done (in reverse chronological order)

[edit] 9/17/07

[edit] Double Digest of AIDA-1 and PCRed rdm-lib

AIDA-1/pet29b+ Double Digestion (37dC for 3 hrs)
Reagent Concentration Volume Total in soln
AIDA-1/pet29b+ DNA~150 ng/uL23.5 uL~3.5 mg
NotI (NEB)10 U/uL2 uL10 U
NheI (NEB)10 U/uL1 uL10 U
Buffer 2 10x5 uL1x
BSA100x.5 uL1x
DEPC water-18 uL-
PCRed rdm lib Double Digestion (37dC for 3 hrs)
Reagent Concentration Volume Total in soln
PCRed insert-21 uL-
NheI (NEB)10 U/uL2.5 uL10 U
NotI (NEB)10 U/uL5 uL
Buffer 2 10x5 uL1x
BSA100x.5 uL1x
DEPC water-16 uL-

Note: NotI is only 50% efficient in Buffer 2 so I also set up a sequential digest (Nick said that one time that anything under 100% for NEB enzymes is unreliable). Also, both the PCRed rdm lib samples above and below came from the first PCR reaction (tube labeled 1 and lane one from the gel).

[edit] Sequential Digest of AIDA-1 and PCRed rdm-lib

AIDA-1/pet29b+ Sequential Digestion Step 1 (37dC for 3 hrs)
Reagent Concentration Volume Total in soln
AIDA-1/pet29b+ DNA~150 ng/uL23.5 uL~3.5 mg
NheI (NEB)10 U/uL1 uL10 U
Buffer 2 10x5 uL1x
BSA100x.5 uL1x
DEPC water-20 uL-
AIDA-1/pet29b+ Sequential Digestion Step 2
Reagent Concentration Volume Total in soln
AIDA-1/pet29b+ (NheI cut) DNA-27.5 uL-
NotI (NEB)10 U/uL1 uL10 U
Buffer 3 10x5 uL1x
BSA100x.5 uL1x
DEPC water-16 uL-
PCRed rdm lib Sequential Digestion Step 1 (37dC for 3 hrs)
Reagent Concentration Volume Total in soln
PCRed insert-21 uL-
NheI (NEB)10 U/uL2.5 uL10 U
Buffer 2 10x5 uL1x
BSA100x.5 uL1x
DEPC water-21 uL-
PCRed rdm lib Sequential Digestion Step 2
Reagent Concentration Volume Total in soln
PCRed insert (NheI cut)-27.5 uL-
NotI (NEB)10 U/uL2.5 uL10 U
Buffer 3 10x5 uL1x
BSA100x.5 uL1x
DEPC water-14.5 uL-

Note: The samples were PCR purified between steps 1 and 2 for both sequential digests.

[edit] 9/15/07

[edit] Miniprepping AIDA-1/pet29b+

Miniprepped the AIDA-1 (~150 ng/uL for all three samples; curves looked good)

[edit] PCRing Random Library

Realized we don't have NotI enzyme so set up a PCR reaction for the random library + AIDA:

Reagent Concentration Volume
AIDA-1/pet29b+ DNA~1.5 ng/uL.5 uL
Primer 1 (MSN27-strong)50 uM3 uL
Primer 2 (MNSIGEM2)100 uM1.5 uL
Platinum PCR supermix-45 uL

Both primers can be found in the -20dC freezer in the small room in a box labeled with green tape (something like "Mike Strong Plasmids for iGEM 2007"). These are primers for the 15mer library.

I used the "rdm lib pcr AIDA" (or something like that) program on the grey machine near the entrance of the big room. The program steps are:

  1. 95dC for 04m00s
  2. 35 loops of:
    1. 95dC for 00m15s
    2. 55dC for 00m30s
    3. 68dC for 02m15s
  3. 68dC for 15m00s
  4. 4dC forever

After the PCR, I ran a gel. The bands looked the correct size and were relatively bright.

[edit] 9/14/07

[edit] Growing Liquid Cultures (AIDA-1/pet29b+)

Grew up 3x4.5mL of AIDA-1 in LB kan from Perry's frozen stock (loc:Perry's box in the -80dC). Tubes were labeled something like "AIDA-1/pet29b+."

[edit] 8/18/07

[edit] Rdm lib + AIDA construction

  1. AP treated 50 µL of AIDA1 cut with NotI/NheI in pet29 with 6 µL AP buffer and 4 µL AP
  2. Clonewelled the samples
  3. Vacufuged and PCR purified the samples
  4. Nanodropped and set up three ligation reactions
    1. PCR digest 1 + cut AIDA
      1. PCR digest - 30 µL of 33.8 ng/µL lyophilized and resuspended in 13.5 µL, used 7 µL
      2. cut AIDA - 47 µL of 37.3 ng/µL lyophilized and resuspended in 9 µL, used 3 µL
    2. PCR digest 2 + cut AIDA
      1. PCR digest - 32 µL of 45.1 ng/µL lyophilized and resuspended in 19.4 µL, used 7 µL
      2. cut AIDA - 47 µL of 37.3 ng/µL lyophilized and resuspended in 9 µL, used 3 µL
    3. PCR digest 3 + cut AIDA
      1. PCR digest - 32 µL of 45.7 ng/µL lyophilized and resuspended in 19.4 µL, used 7 µL
      2. cut AIDA - 47 µL of 37.3 ng/µL lyophilized and resuspended in 9 µL, used 3 µL

[edit] Re-electroporating tagged Single Cells + T02 his

I had to miniprep more JT because we used up the plasmid preps from last time

  1. JT DNA into AIDA his cells (from Steph's fridge liquid cultures)
  2. JT DNA into AIDA+strep cells (from Steph's fridge liquid cultures)
  3. RACE DNA into AIDA+strep cells (from Steph's fridge liquid cultures)
  4. RACE DNA into AIDA+strep cells (from Steph's fridge liquid cultures)
  5. AIDA+his DNA (from Mike's iGEM box) into T02 cells

Plates should be in incubator

[edit] Checking Tagged Receivers

  • Round 1
    • Colony PCRed the RAE+his, RAE+strep, and T02+strep (the T02+his plate was empty).
    • Unfortunately, there are only SybrSafe E-gels left, so no gel picture. Gel summary below (basically RAE+strep 2 and T02+strep 2-5 worked)
Well Contents Band Length Actual Length Correct?
11 kb+---
2RAE+his 110002000No
3RAE+his 2none2000No
4RAE+his 3none2000No
5RAE+strep 1none2000No
6RAE+strep 220002000Yes
7RAE+strep 3none2000No
8T02+strep 11100ish2000No
9T02+strep 2none2000No
10T02+strep 320002000Yes
11T02+strep 420002000Yes
12T02+strep 520002000Yes
  • Round 2
    • Basically I redid RAE+his 1-3 and then streaked three more colonies to get RAE+his4-6. I also checked RAE+strep 2 and T02+strep 3-5 with the T7 promoter/terminator primers. Note I have two wells of RAE+strep 2 because I distrust (is that a word?) colony PCR.
    • Results were good. The RAE_strep 2 and T02 strep3-5 check out completely. It appears that RAE+his 3 also checks out. I've set up the T7 PCR reaction for that have it sitting in the black PCR machine on Stephanie's bench.
Well Contents Band Length Actual Length Correct?
11 kb+---
2RAE+his 1450 (faint)2000No
3RAE+his 2450 (faint)2000No
4RAE+his 320002000Yes
5RAE+his 4none2000No
6RAE+his 5none2000No
7RAE+his 6none2000No
8RAE+strep 2A1700ish1500Yes
9RAE+strep 2B1700ish1500Yes
10T02+strep 31700ish1500Yes
11T02+strep 41700ish1500Yes
12T02+strep 51700ish1500Yes

[edit] Other

  • Finally streaked those liquid cultures of the constructs we sequenced.
  • Streaked the liquid culture of pAC-luxGFP (Voigt construct)
  • miniprepped the pAC-luxGFP
  • poured several LBamp+kan plates. They should be labeled with black sharpie on the broad circular face of the plate.

[edit] 8/14/07

[edit] Colony PCR of RACE (new)

RACE #1-3 are correct.

Gel

[edit] 8/13/07

[edit] Fluorescent Microscopy of RACE

I picked from six samples from three plates of RACE and dipped into 10 µL of PBS:

  • RACE 37dC (new) - the new RACE transformation plate
    • Center of the colony (fluorescent)
    • Edge of the colony (nonfluorescent)
  • RACE 37dC (old) - the RACE plate the old transformation
    • fluorescent cells
    • nonfluorescent cells
  • RACE - the original RACE transformation plate
    • Center of the colony (fluorescent)
    • Edge of the colony (nonfluorescent)

Microscopy Results:

  • For the most part, the expected nonfluorescent cells are nonfluorescent and the fluorescent cells are fluorescent. However, it seems that the original plates are exhibiting stronger fluorescence than the new transformation plates which is opposite of what Nick, Harris, Bill and I observed in the transformation plates
  • I need to fix my protocol for preparing slides because I have problems with the ends of my slides drying in and some of my samples were very dilute.
  • These RACE cells didn't have the filament problems like the last RACE I grew. I'm going to retry the RACE+OHHL and RACE overnight to see if I can get some positive results.

Fluorescent Microscopy Pics

[edit] 8/10/07

[edit] Colony PCR of JT + confirm Master Plate

JT's are whacko, the Master Plate constructs are correct. We need to re-transform JT into BL21.

Gel

[edit] Frozen Stock Part 1

Perry and I made frozen stocks of some stuff.

[edit] 8/09/07

[edit] Mixed Cultures with same OD

I had a culture of S23I07 diluted to OD=0.057 and a culture of RAE diluted to an OD=0.059. I made a 1:1, 10:1, and 100:1 combined culture (with more of the RAE). I also had a culture of RACE that I induced with ~1 mM.

I grew these for about three hours and then checked them in the plate reader and the fluorescent microscope.

Plate Reader Data 1
Sample OD GFP RFP
RACE+OHHL0.71679.196179.94
1:10.866161.96257.00
10:10.729314.73204.50
100:10.405477.33216.94

Fluorescent Microscope Results #1:

  • Not too much to say here, it seems that the cells haven't grown enough because we don't see much fluorescene yet and the OD's are still pretty low
  • One thing of note: the RACE is abnormally filamentous. Something is wrong...

Fluorescent Microscopy Pics 1

I grew the samples for another two hours and then checked them again in the plate reader and the fluorescent microscope.

Plate Reader Data 2
Sample OD GFP RFP
RACE+OHHL1.23580.495165.18
1:11.278205.86444.09
10:11.234414.76537.42
100:11.047670.86234.11

Fluorescent Microscope Results #2:

  • The RFP fades RIDICULOUSLY fast. The fluorescence fades to black in a couple of seconds.
  • The GFP in the mixed cultures is pretty good, the RFP is almost invisible. This could be the result of the RFP folding time and the fact that the RFP fades RIDICULOUSLY fast. Seriously, like, Nick and I basically had to say "1...2...3... GO!" to make sure we got the picture on time

Fluorescent Microscopy Pics 2

[edit] 8/08/07

I took plate reader data of the swab inoculated liquid cultures:

Sample Spectro. OD Plate OD GFP RFP
S23.I07+R62.A20.E402.2932.047249.345706.1
B151.7821.567108.47158.64
S23.I07+T022.1711.956616.056045.8
OHHL+T021.8121.76263.01168.93
I222.0691.91910101187.39
OHHL+R62.A20.E401.9231.821770.6163.89
R62.A20.E401.891.735166.23175.59
Blank LB-0.09677.822165.43

I also checked the cultures under the fluorescent microscope:

  • The S23I + RAE sample showed a lot of red colonies and a very small number of green colonies.
    • Maybe the red colonies are just much better competitors? It could also be because I didn't start with equal OD's. I'm going to redo this tomorrow.
  • The RAE works surprisingly well. By itself, it has a pretty good OFF and the induced with OHHL was brightly fluorescent.

Fluorescent Microscopy Pics

[edit] 8/07/07

[edit] Colony PCR for Master Plate

Colony PCR is a stupid protocol

Gel

[edit] 8/06/07

[edit] Colony PCR for Master Plate

Whaaa?

Gel

[edit] 8/03/07

[edit] Colony PCR of luxpR Senders and Receivers

Whaaa?

Gel

[edit] 8/02/07

[edit] Colony PCR of luxpR JT + mCherry parts

The mCherry colonies were not red T_T.

Gel

[edit] 8/01/07

[edit] Colony PCR of mCherry Part

Most of the streaks looked good

Gel

[edit] 7/31/07

[edit] Colony PCR of luxpR Round 2 Take 1

Colony PCR'ed the transformation plates from last night. The transformation plates looked very good, with lots of colonies.

Gel

[edit] 7/30/07

[edit] luxpR Construction Round 2

Miniprep, Digest, Dephosphorylate, Clonewell, Ligate, Transform:

The only difference is that the Minipreps turned out a lot better, giving nanodrop values of >130 ng/µL. Also, I stupidly transformed into Top10F' instead of Top10.

[edit] 7/29/07

[edit] Colony PCR of luxpR Round 1 Take 2

I colony PCR'ed the colonies from the transformation plates from last night. The plates were a little better, with around 10 colonies each. I only got *i think* the C0061<I13504 to work. Also, the five bands that should be R0062<A340620 are all about the size of R0062 so I'm going to redo the construction tomorrow.

Gel

[edit] 7/28/07

[edit] Colony PCR of luxpR Round 1 Take 1

Unfortunately, there were like no colonies. Meaning:

So I colony PCR'ed them and then froze the result (no gel running today, folks).

[edit] Religating and Transforming

I re-ligated and transformed some leftover DNA from last night:

Finally, I transformed the constructs into Top10 cells.

[edit] 7/27/07

[edit] Clonewell, Vacufuge, Ligate, Transform

I clonewelled, vacufuged, and then ligated the following:

Finally, I transformed the constructs into BL21 cells.

[edit] Dephosphorylating the Vectors

I dephosphorylated the R0062 and C0061 with Antarctic Phosphotase and then heat inactivated them for 10m at 65dC.

[edit] Digesting for Double luxpR

I digested the following for 99:99 at 37dC:

Part DNA Enzyme 1 Enyzme 2 Buffer 2 BSA
R006220 µL1 µL SpeI1 µL PstI2.5 µL0.5 µL
A34062020 µL1 µL XbaI1 µL PstI2.5 µL0.5 µL
C026120 µL1 µL SpeI1 µL PstI2.5 µL0.5 µL
E024020 µL1 µL XbaI1 µL PstI2.5 µL0.5 µL
I1350420 µL1 µL XbaI1 µL PstI2.5 µL0.5 µL

[edit] Sequencing the C0261

I prepared and sent C0261 for sequencing.

[edit] Miniprep for Double luxpR

Perry and I miniprepped:

in order to build the luxpR construct.

[edit] 7/26/07

[edit] FACS

Perry and I diluted down overnight samples and grew them in an incushaker from 9:30-12:30. The numbers indicate OD right before we resuspended in PBS. We tried to resuspend so that the final OD was around 0.003.

  • JT
    • 1:10 (.555)
    • 1:100 (.141)
    • 1:1000 (.02)
    • 1:10000 (.000-.001)
  • T02&S23I07 1:1000 (.03)
  • T02&J23039 1:1000 (.03)
  • F20I07&S23E40 1:1000 (.075)
  • J04450 1:100 (.064)
  • I13522 1:100 (.62)
  • B0015 1:100 (.061)

Media:072707facsgx+pt.zip

[edit] Overnight Plate Reader Growth Curve Results

I set up a plate reader as follows. The numbers after the label indicate starting OD. Note: Our plate reader seems to have trouble reading OD's around 0.1, so I'm not completely sure if the 0.1 values are accurate. I took the lower OD readings in the OD machine in the small room, so I'm pretty confident about those.

123456789101112
AT02
0.106		T02
0.106		T02
0.106		J39
0.106		J39
0.106		J39
0.106		S23I07
0.106		S23I07
.106		S23I07
0.106		S23E40
0.105		S23E40
0.105		S23E40
0.105
BS08E40
.109		S08E40
.109		S08E40
.109		S08I07
0.11		S08I07
0.11		S08I07
0.11		BLANK LBBLANK LBBLANK LBBLANK LBBLANK LBBLANK LB
CT02
0.002		T02
0.002		T02
0.002		J39
0.001		J39
0.001		J39
0.001		S23I07
0.002		S23I07
0.002		S23I07
0.002		S23E40
0.002		S23E40
0.002		S23E40
0.002
DS08E40
.003		S08E40
.003		S08E40
.003		S08I07
0.004		S08I07
0.004		S08I07
0.004		BLANK LBBLANK LBBLANK LBBLANK LBBLANK LBBLANK LB
ET02
0.043		T02
0.043		T02
0.043		J39
0.038		J39
0.038		J39
0.038		S23I07
0.040		S23I07 0.040S23I07
0.040		S23E40
0.041		S23E40
0.041		S23E40
0.041
FS08E40
.042		S08E40
.042		S08E40
.042		S08I07
0.045		S08I07
0.045		S08I07
0.045		BLANK LBBLANK LBBLANK LBBLANK LBBLANK LBBLANK LB

Results:

  • Starting at OD~0.1 or OD~0.04 it took the samples around 2 1/2 hrs to read OD~0.3 and around a total of 6 hrs to hit stationary phase
  • Starting at OD~0.003, it took the samples around 4 hours to reach OD~0.3 and around a total of 7 hours to hit stationary phase
  • There didn't seem to be a big difference between starting at OD~0.1 vs OD~0.04. Of course, this could be due to the fact that I took the OD reading for the OD~0.1 samples in the plate reader, which is inaccurate in that range.
  • Overall, the samples grew at pretty much the same rate. There appears to be at most a 45-60 min lag between different constructs, but this could be due to variations in starting OD.
  • The blank LB samples were interesting. They were pretty much flat and low until around 8 hrs in, then they grew exponentially to around OD~1. This seems to indicate that there were very low levels of contamination in these LB wells. I added LB+amp to these wells, so the contamination is probably coming from our own constructs. I don't know if this contamination was present when I loaded the wells or somehow there was cross-well contamination during the reading period.

For all the graphs+raw data: Media:072607platereadergx.xls

[edit] 7/25/07

[edit] Sorting through Sequences

I went through all the sequences we have done and compiled the results

Sequencing Results

[edit] Miniprepping, Nanodrop, and Sequencing S03608.I13507

I miniprepped and sent S03608>I13507 for sequencing. Elapsed time: 20 minutes.

Sequencing Results

[edit] Colony PCR of various parts

Perry and I colony PCR'ed the following parts:

Gel

[edit] 7/24/07

[edit] Liquid Cultures

Perry and I grew liquid cultures of several things:

[edit] Mike's Agar Plate Experiment

Perry and I carried out a preliminary test of Mike's Plate Reader Experiment. Protocol:

  1. Pipet 200 µL of overnight cultures of T9002 and spread onto LB agar plates.
  2. Let the plates sit at room temperature to allow the bacteria to soak in. (We let it sit for about 20 minutes)
  3. Pipet in 10 µL of one of each of the following into the middle of each plate.
    1. Overnight culture of S03623 > I13507
    2. Overnight culture of S03623 > I13507
    3. Overnight culture of S03608 > I13507
    4. Overnight culture of S03608 > I13507
    5. 2 µM OHHL

We did duplicates because we took from two different overnight cultures, one that was obviously red and one that was not obviously red.

[edit] Ligating and Transforming the RP-JT and FluorSwitch

Perry and I performed the following ligations and transformations:

into BL21

We decided not to do the other ligations and transformations because we found out that we already have a S03623 <E0240 and we were colony PCR'ing a S03608<E0240. Finally, because the J23039 >T9002 miniprep and clonewell sucked, we were reluctant to move forward on that ligation.


[edit] Clonewell for RP-JT and FluorSwitch

I clonewelled and vacufuged all the digests I did earlier in the day. The band for J23039 >T9002 was almost invisible.

[edit] Sequencing the F2620 and E0240

I prepared a sequencing order for F2620 and E0240.

Sequencing Results

[edit] Dephosphorylate for RP-JT and FluorSwitch

I dephosphorylated the following:

with Antarctic Phosphatase for 1 hr at 37dC and then heat inactivated for 10m at 65dC.

[edit] Digest for RP-JT and FluorSwitch

I digested the following for 99m99s.

Part DNA Enzyme 1 Enzyme 2 Buffer 2 BSA
R0011 <P014021 µL 1 µL EcoRI1 µL XbaI2.5 µL 0.5 µL
R0051 <P014021 µL 1 µL EcoRI1 µL SpeI2.5 µL 0.5 µL
J23039 >T900221 µL 1 µL EcoRI1 µL SpeI2.5 µL 0.5 µL
S0362321 µL 1 µL XbaI1 µL PstI2.5 µL 0.5 µL
S0360821 µL 1 µL XbaI1 µL PstI2.5 µL 0.5 µL
E024021 µL 1 µL SpeI1 µL PstI2.5 µL 0.5 µL
F262021 µL 1 µL EcoRI1 µL SpeI2.5 µL 0.5 µL
I1350721 µL 1 µL EcoRI1 µL XbaI2.5 µL 0.5 µL

[edit] Miniprep (RPJT+FluorSwitch)

I miniprepped the samples that were grown last night:

All the nanodrop data was good (about 150 ng/µL with excellent curves), except for the J23039 >T9002 (50ng/µL)

[edit] 7/23/07

[edit] Colony PCR of RP

I colony PCR'ed the R-P constructs that didn't work out the first time:

Gel

[edit] Growing Liquid Cultures

I grew liquid cultures:

[edit] 7/22/07

[edit] Colony PCR of R-PJT

Gel

[edit] 7/21/07

[edit] Clonewell RP and JT

I clonewelled the following things (some are Perry's samples):

and then vacufuged them.

[edit] Dephosphorylating JT

I dephosphorylated JT with Antarctic Phosphotase (1 hr 37dC) and then heat inactivated at 65dC for 10 min.

[edit] 7/20/07

[edit] Clonewell and Vacufuging the RP and JT parts

I clonewelled the leftovers from the RP and JT digests and then vacufuged and resuspended in 20 µL of nuclease free water. This was done in order to try rebuilding the constitutive tetR+JT construct.

[edit] 7/19/07

[edit] Liquid Cultures of 2 cell system and RFP+JT

I grew liquid cultures of the 2 cell system and the RFP+JT in preparation for plate reader experiments tomorrow.

[edit] Sequencing the RFP+JT part

I prepared the RFP+JT part and sent it for sequencing.

Label Sample Primer
AV01J04450-JTVF2
AV02J04450-JTVR
AV03jSLOPDZ2BVF2
AV04jSLOPDZ2BVR
AV05jSLOPDZ1BVF2
AV06jSLOPDZ1BVR
AV07JhisGFPmekVF2
AV08JhisGFPmekVR

Sequencing Results

[edit] Colony PCR of the tetR+JT constructs Part II

I chose only non-fluorescent colonies (by checking the colonies under the fluorescent microscope) for this colony PCR.

Results: None of the colony PCR's gave the correct size DNA piece.

Gel

[edit] Miniprep the RFP+JT

I miniprepped the RFP+JT sample (originally for transformation in Top10 because BL21 expresses LacI, but we decided that we liked the idea of inducible RFP).

[edit] 7/18/07

[edit] Liquid Cultures

I grew liquid cultures of the "correct" RFP+JT colony resulting from the colony PCR.

[edit] Colony PCR of the tetR+JT and RFP+JT constructs

I colony PCR'ed the teR+JT and RFP+JT constructs.

Results: Only one colony seemed correct. It was one of the constitutive RFP+JT parts.

Gel

[edit] 7/17/07

[edit] Ligation and Transformation of tetR-JT and RFP-JT constructs

I Roche ligated the following:

and then transformed them into BL21.

Ligation Protocol Transformation Protocol

[edit] Clonewell and Vacufuge of the R-P constructs

I clonewelled and vacufuged the R-P constructs and the JT. Unfortunately, I forgot to include the JT with the initial clonewell, so I ended up having to fish the very nasty clonewell out of the garbage (there was a piece of gel on it and chunks of gel were stuck in some of the wells). I used the last two wells (which were thankfully somewhat clean) for ladder and the JT. Unfortunately I ran into a few problems. First, the JT is about 2800 bp and the plasmid is about 2000 bp. The two bands on the clonewell were pretty close and it was difficult to separate the two bands sometimes. Second, the gel stopped running after a while. I noticed bubbles forming along the edges of the gel and whenver I pressed "Go," the gel would make an unhappy beeping noise and blink yellow, so I just stopped running the clonewell

Protocol

[edit] Dephosphorylating the R-P constructs

Construct DNA AP Buffer AP
R0011 <P0340 colony #240 µL4.7 µL2 µL
R0051 <P0140 colony #142 µL4.9 µL2 µL
R0051 <P0340 colony #136 µL4.2 µL2 µL
R0052 <P0140 colony #2 Lower Right40 µL4.7 µL2 µL
R0052 <P0340 colony #1 Upper Right40 µL4.7 µL2 µL

I then incubated at 37dC for 1 hour and heat inactivated at 65dC for 10 minutes.

Protocol

[edit] Sequencing the R-P constructs

I prepared and sent off reaction mixes. A summary of the order below:

Label Construct Primer
AV01J-hisGFPmek_5_1VF2
AV02J-hisGFPmek_5_1VR
AV03J_hisGFPmek_8_1VF2
AV04J_hisGFPmek_8_1VR
AV05R0011 <P0340 colony #2VF2
AV06R0011 <P0340 colony #2VR
AV07R0051 <P0140 colony #1VF2
AV08R0051 <P0140 colony #1VR
AV09R0051 <P0340 colony #1VF2
AV10R0051 <P0340 colony #1VR
AV11R0052 <P0140 colony #2 Lower RightVF2
AV12R0052 <P0140 colony #2 Lower RightVR
AV13R0052 <P0340 colony #1 Upper RightVF2
AV14R0052 <P0340 colony #1 Upper RightVR

Sequencing Results

[edit] Digesting JT and R-P constructs

I digested the following for 99:99 minutes:

Construct DNA Enzyme 1 Enzyme 2 Buffer 2 BSA
R0011 <P0340 colony #240 µL1 µL XbaI1 µL PstI4.7 µL.5 µL
R0051 <P0140 colony #133 µL1 µL SpeI1 µL PstI4.0 µL.5 µL
R0051 <P0340 colony #135 µL1 µL SpeI1 µL PstI4.2 µL.5 µL
R0052 <P0140 colony #2 Lower Right30 µL1 µL SpeI1 µL PstI3.6 µL.5 µL
R0052 <P0340 colony #1 Upper Right33 µL1 µL SpeI1 µL PstI4.0 µL.5 µL
J23039 <T900233 µL1 µL SpeI1 µL PstI4.0 µL.5 µL

[edit] Miniprep and Nanodrop of JT and the R-P constructs

I miniprepped the following parts:

The "5" and "8" were Perry's samples. Note that the R0052 <P0340 colony #1 Lower Right is missing. That is because the overnight culture didn't seem to grow. After centrifuging several times, there was still no pellet, so I just tossed that sample out.

The Nanodrop results (after Nanodropping, I made the following dilutions for sequencing samples):

Construct Concentration Dilution µL of DNA µL of Water
R0011 <P0340 colony #297.6 ng/µL1 in 21010
R0051 <P0140 colony #1173.2 ng/µL1 in 4515
R0051 <P0340 colony #1