Spring 2006 pCMV-Tag2A / hDlg work: Difference between revisions
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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. | 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. | ||
[[Image:03- | [[Image:03-10 PCR pur PT.jpg]] | ||
Lane 1: 1 kb ladder | Lane 1: 1 kb ladder | ||
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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. | 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. | ||
[[Image:03-10 PCR pur-2 PT.jpg]] | |||
Lane 1: 1 kb ladder | Lane 1: 1 kb ladder | ||
Revision as of 21:49, 10 March 2006
Notes from fall 2005 have been archived into "Perry, fall 2005"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
