Team:Heidelberg/Templates/MM week13p
From 2013.igem.org
Contents |
2013-07-22
- transform BBa_I746200 in pSB1AK3 (Spring 2012 Distribution Plate 2 Well 15E) in TOP10, plate on Amp
- transform BBa_J04450 in pSB3C5 (Spring 2012 Distribution Plate 1 Well 3C) in TOP10, plate on Cm
2013-07-23
- pick TOP10-BBa_I746200 colony, inoculate LB+Kan, grow at 37°C
- pick TOP10-BBa_J04450 colony, inoculate LB+Cm, grow at 37°C
- after 12 hours: add new antibiotics to medium, continue growing at 37°C
2013-07-24
- Prepare glycerol stocks
- make miniPreps: 11.1 ng/µl (BBa_J04450), 18 ng/µl (BBa_I746200)
2013-07-25
- run PCRs (using Q5, 20 µl total volume) using 4 ng of each template (pLF03 miniPrep from 2013-06-11, 21 ng/µl), sfp from Konrad's BAP1 plate:
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 (f1,f2,f3,f5,f6) / 300 (f4) |
35 | 98 | 5 |
66 (f1,f2) / 60 (f4) / 61 (f3,f5,f6) | 10 | |
72 | 120 (f1,f2,f3,f5,f6) / 30(f4) | |
1 | 72 | 600 |
1 | 12 | inf |
- did not manage to get f3, f4, f6
- repeat: add 1 µl DMSO to reactions:
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 |
35 | 98 | 5 |
56 (f4) / 57 (f3,f6) | 30 | |
72 | 150 (f3,f6) / 45(f4) | |
1 | 72 | 600 |
1 | 10 | inf |
- use pLF03 miniPrep from 2013-06-11, 27 ng/µl, repeat for f3, f6 (with 1 µl DMSO):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 |
12 | 98 | 10 |
62 ↓0.5°C | 10 | |
72 | 150 | |
23 | 98 | 10 |
58 | 10 | |
72 | 150 | |
1 | 72 | 600 |
1 | 10 | inf |
2013-07-26
- run PCR of f3, f6 with Phusion Flash (using pLF03 miniPrep from 2013-06-11, 21 ng/µl), without DMSO:
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
35 | 98 | 1 |
65 | 5 | |
72 | 75 | |
1 | 72 | 600 |
1 | 10 | inf |
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
5 | 98 | 1 |
65 | 5 | |
72 | 75 | |
30 | 98 | 1 |
72 | 75 | |
1 | 72 | 600 |
1 | 10 | inf |
- inoculate 10 ml LB+Amp with TOP10-pLF03, grow for 36h at 37°C, add Amp every 12 hours
2013-07-28
- make miniPrep -> very low yield (ca. 1-2 ng/µl)
- inoculate 6 ml LB+Amp with TOP10-pLF03, grow at 37°C