Spring 2006 pCMV-Tag2A / hDlg work

Notes from fall 2005 have been archived into "Perry, fall 2005"

Table 1: Composition of the hDlg constructs

Construct	Parts
LS25G	P1 + P2
LS35G	P1 + P3
LS325G	P1 + P4 + P5
LS254G	P1 + P6 + P7 + P8
LS354G	P1 + P4 + P9 + P8

Table 2: Composition and construction of parts

Parts	Domains	Template	Primers
P1	L27	F11	L27S + L27A
P2	SH3-I2-I5-GK	SG25	SH3S + GKA
P3	SH3-I3-I5-GK	SG35	SH3S + GKA
P4	SH3-I3	SG35	SH3S + I3A
P5	I2-I5-GK	SG25	I2S + GKA
P6	SH3	F11	SH3S + SH3A
P7	I2-I5-I4	I2-I5-I4/Topo	I2S + I4A
P8	GK	F11	GKS + GKA
P9	I5-I4	I2-I5-I4/Topo	I5S + I4A

3/3/06

Transformed DH5alpha competent cells (50 ul aliquots) with pcMV-Tag2A vectors. The stock received was 1 ug/ul, used 1 ul for transformation (did two identical ones), 2.5 ul pUC19 for (+) and nothing for (-). On ice for 30 minutes. Heat shock 42 degrees Celsius for 25 seconds. On ice for 2 minutes. Added 950 ul prewarmed SOC, and shook at 225 rpm, 37 degrees, for 1 hour. (followed protocol from DH5alpha product information, except for heat shock step)

Transformed TOP10 competent cells with hDlg-cloned Topo vectors SG25, SG35, and F11. Diluted stock vector 1:10, used 5 ul for transformation, 2.5 ul pUC19 for (+), nothing for (-). On ice for 30 minutes. Heat shock 42 degrees Celsius for 30 seconds, on ice for 2 minutes. Added 250 ul room-temp SOC, and shook at 225 rpm, 37 degrees for 1 hour. (followed previous MCB100 protocol)

Plated bacteria with controls and Topo vectors on carb plates, bacteria with pCMV vectors on kan plates, with glass beads under burner. Placed in 37 degree incubator at 6 pm.

3/4/06

Transformations didn't go great, so Yi-an reperformed them.

3/5/06

hDlg transformations from yesterday didn't show identifiable colonies, more a smear of nothing. pCMV transformations from yesterday produced a few colonies. hDlg transf from Friday were overgrown in SG35 and F11; must have used too much vector to transform.

Prepared three 200 ml LB liquid cultures from single colonies of bacteria transformed with hDlg F11, SG25, and SG35, with 200 ul ampicillin (50 mg/ml); and one 200 ml, pCMV, with 200 ul kanamycin (50 mg/ml). put in shakers around 9:30 pm, set at 300 rpm and 37 degrees Celsius. also streaked onto new plates.

3/10/06

Yi-an already performed the midipreps and PCRs. Today I PCR purified P5-9. Before purification, I took out 10 ul fromeach and added 10 ul water for 1.2% E-gel. I measured the volume left and added 5 volumes Buffer PB to each PCR product. P5 had 20 ul, and I added 100 ul PB; P6, 35, 175; P7, 35, 175; P8, 30, 150; P9, 35, 175. I proceeded with PCR purification, eluting with 30 ul EB. I took out 10 ul from each purified product and added 10 ul water for E-gel. I also took out 10 ul of 1 kb ladder and of 2 log ladder, and added 10 ul water for E-gel.

Lane 1: 1 kb ladder

Lane 2: P5 before

Lane 3: P5 after

Lanes 4-11: P6-9 before and after

Lane 12: 2 log ladder

P5 and P6 have the right lengths.

Setup PCRs for P7-9 again, but this time with undiluted I2-I5-I4/Topo for 7 and 9. Repeated E-gel with 10 ul before and after PCR purification. The volume left for PCR purification was 37 ul in each, and I added 185 ul PB to each.

Lane 1: 1 kb ladder

Lane 2: P7 before

Lane 3: P7 after

Lanes 4-7: P8 and P9 before and after

Lane 8: 2 log ladder

Performed Topo vector ligation using P5, P6, and P8, with 4 ul PCR product, 1 ul Salt solution, 1 ul Topo vector. Incubated room temp 5 min, transferred into tube of chemically competent Top10 cells. Split one 60 ul tube into two 30 ul tubes, one for untreated (-) and one for pUC19-transformed positive (+). On ice for 20 minutes, heat shock at 42 degrees Celsius for 30 seconds, one ice for 2 minutes, added 250 ul SOC media to each tube, shook at 37 degrees Celsius for 1 hour, plated w/ carb.

3/13/06

Transformed one tube of Top10 with 2 ul of original SG35-cloned vector, left one tube untreated for negative. Used 350 ul SOC, and plated a 50, 100, and 200 ul plate from each tube.

Prepared 2 identical double digests of pCMV midiprep: 5 ul pCMV (~213 ng/ml), 2 ul BamHI buffer (10X), 0.2 ul BSA (100X), 0.5 ul BamHI, 0.5 ul XhoI, 11.8 water. Left in PTC-100 at 37 degrees for 6 hours, 80 degrees for 20 minutes, 4 degrees indefinitely.

3/14/06

Ran digest on gel.

Lane 1: 1 kb ladder

Lane 3: pCMV digest #1

Lane 5: pCMV digest #2

The upper band is the correct size for linear vector, and the lower band is probably supercoiled undigested vector.

Performed 2 parallel QIAEX II gel extractions, for DNA >4kb. Preweighed centrifuge tubes at 1.016 and 1.030 g, then weighed with gel slices at 1.108 and 1.161 g. Used 300 and 390 ul QX1, 30 ul QIAEX II each b/c of strong bands, but I forgot to add 2 volumes of water to each. After 50 degree incubation, I added 10 ul 3M sodium acetate to each because of a pinkish color which then turned yellow. Air dried over 30 minutes until about half of each pellet turned from clearish gray to white. Eluted each twice with 20 ul water. Ended up with 2 tubes of ~35 ul elution. Froze one tube, used other tube for a redigestion.

Prepared 3 digests.

1. New digest of pCMV, same as yesterday except with 3 ul DNA and 13.8 ul water.

2. A negative control digest with 3 ul DNA, no enzyme, and 14.8 ul water.

3. A redigest of the DNA that was gel extracted today, using 35 ul gel extraction, 5 ul BamHI buffer, 0.5 ul BSA, 1.25 ul BamHI, 1.25 ul XhoI, and 7 ul water, for a 50 ul digestion.

Ran digest at 37 degrees for 12 hours, 80 degrees for 20 minutes, and 4 degrees indefinitely.

Had to pause the digestion at 3 hours and use PTC machine for annealing BB linkers. 5 ul BBCS1 (for 50 pmol from 10 uM), 5 ul BBCS2, 5 ul T4 ligase buffer (10X), 0.5 ul T4 kinase, 34.5 ul water, at 37 degrees for 30 minutes, 65 degrees for 20 minutes, 94 degrees for 1 minute, let cool to room temperature.

Left digests out at room temp. Returned to PTC after linkers were done and completed 9 more hours of 37 degree incubation.

Helped Yi-an with gel extraction of PCR products. Repeated steps for QUIAEX II protocol, only used 3 volumes of QX1, 10 ul of QIAEX II, added 10 ul sodium acetate two times until yellow. Eluted twice with 20 ul water.

Performed Topo cloning with 1 ul vector, 1 ul salt solution, 4 ul gel-purified PCR product. Transformed Top10 cells and plated onto new amp plates which Alain poured.

3/15/06

Yi-an ran gel of digests for me.

Lane 1: 1 kb

Lane 3: Undigested vector

Lane 5: New digest with 3 ul midiprep

Lane 7: Redigest of gel extracted DNA

Lane 9: Gel extracted DNA.

The 2nd band appeared again, and did not match up with supercoiled undigested plasmid. So we suspect that this is an actual insert from the plasmid. pCMV-Tag2 control plasmid (for transfection control) includes an Luciferase insert. Cut out bands from Lanes 5, 7, and 9, and froze.

Yi-an performed two transformations using DNA from tubes labelled pCMV-Tag2A and pCMV-Tag2control.

Topo cloning/transformation with the gel-purified PCR products produced colonies. Yi-an prepared inoculations and made new streaks from four different colonies on each transformation plate. (P1-, P2-, P3-, P4-, P5-, P6-, P8-1 through 4)

3/16/06

Alain spun down pellets from inoculations. I performed minipreps. I spilled the Buffer P1 resuspension of the Part 2-2 pellet on the bench, pipet it off the bench and back into the tube, and proceeded normally with the miniprep. I prepared EcoRI digests from all the minipreps: 10 ul DNA, 2.5 ul EcoRI buffer, 0.25 ul BSA, 0.625 ul EcoRI, 11.625 water, for a 25 ul digest. Left in PTC at 37 degrees for 12 hours, 80 degrees for 20 minutes, 4 degrees indefinitely.

Ran gel of digests.

Lane 1: 1 kb ladder

Lanes 2-5: P1-1 through 4

Lanes 6-9: P2-2 through 4

Lanes 10-13: P3-3 through 4

Lanes 14-15: P4-1 and 2

Lane 1: 1 kb ladder

Lanes 2-3: P4-3 and 4

Lanes 4-7: P5-1 through 4

Lanes 8-11: P6-1 through 4

Lanes 12-15: P8-1 through 4

The bands that appeared for P1, 4, 5 are the correct size. No bands appeared for P2 and 3. The bands for P6 are the incorrect size.

3/19/06

Repeated pCMV transformation using Tag2A and Tag2, with DH5alpha competent cells. Followed the issued protocol. Plated 200 ul from the 1ml SOC mixtures and refrigerated the rest.

3/20/06

Inoculated Tag2 and Tag2A from colonies (ran toothpick across several colonies from each plate) with 3ml LB, 2ul kan. Ran Yi-an's digests on gel. They were EcoRI digests of DNA miniprepped from inoculations of four more colonies from the latest plates of transformations with Topo vectors cloned with PCR products.

Lane 1: 1 kb plus ladder

Lanes 2-5: P2-5 through 8

Lanes 6-9 P3-5 through 8

Lanes 10-13: P5-5 through 8

Lanes 14-17: P6-5 through 8

Lane 18: 1 kb plus ladder

3/24/06

Gel extracted from slices that Alain excised yesterday of digested pCMV Tag2A. One slice came from two lines, and the other slice came from one lane. Performed two parallel extractions, eluted each twice with 20ul water, and collected elutions into one tube.

Performed three ligation/transformations using digested pCMV Tag2A vector which I had just gel-extracted, and BB linker insert created 3/14/06: (1) 5ul insert, 5ul vector, (2) 2.5ul insert, 7.5ul vector, (3) 7.5ul insert, 2.5ul vector. But I absentmindedly used Top10 cells. Alain realized my mistake right after addition of SOC. Great. Repeated the three ligation/transformations using DH5alpha cells.

Purified product from Alain's PCR reactions #1-6 from yesterday. Eluted with 30ul EB, except accidentally used 50ul for #1, and lost a drop from the PB/PCR mixture from #6.

Alain first PCRed out I2I5I4 from a cDNA library using primers C and BL16. Then he PCRed the product with our primers. #1 and 2 are same PCR reaction with primers I2S and I4A. #3 and 4 are I5S and I4A. #5 and 6 are I3S and I4A.

TOPO cloning: 4ul purified PCR product, 1ul salt solution, 1ul Topo vector. Used PCR purified products #2, 3, 5. Transformation with Top10 cells, 250ul SOC, on carb plates.

3/25/06

Transformation plates of Topo clonings grew well, almost too well. An e-gel Alain ran of the PCR purifications of #2, 3, 5 showed no bands. We suspect that the cDNA plasmids somehow got through the PCR purifications. We plan to do only gel isolations from now on.

Transformation plates of pCMVTag2A-BB linker did not grow. The DH5alpha protocol calls for 1mL initial growth mixture, plating 200ul, and refrigerating the rest. I plated the remaining ~800ul onto new plates and left in incubator. Alain will take them out on Monday.

4/4/06

Prepared PCR reaction with 44ul Platinum PCR Supermix, 2ul P1TagR primer (10uM), 2ul L27TagS primer (10uM), and 2ul P1-1a miniprep (1:100). Used BB4 program in PCR machine #6.

Performed midiprep of pCMVTag2A. Eluted with 500ul water, yielded 424 ng/ul. Used 2ul in a digest: 14.8ul water, 2ul DNA, 0.2 BSA (100X), 2ul BamHI buffer, 0.5ul BamHI, 0.5ul XhoI. Ran digest at 37 degrees for 12 hours, 80 degrees for 20 minutes, 4 degrees indefinitely.