Team:Heidelberg/Templates/MM week13
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Nils.kurzawa (Talk | contribs) m (Created page with " == 2013-07-22 == [[File:BAP1-colony-PCR2013-07-22.png|100px|thumb|right|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid c...") |
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== 2013-07-22 == | == 2013-07-22 == | ||
- | [[File: | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-22.png|100px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2). Lane 1: NEB 2-log; lanes 2,4: BAP1-pLF03 (control); lanes 3,5: colony-PCR. Lanes 2,3: primers IK01+IK03; lanes 4,5: primers RB43+RB44]] |
- | [[File: | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-22_2.png|100px|thumb|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers RB43+RB44. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR; lane 4: streaked BAP1 by Konrad]] |
* no positive PCR of colony yet: run colony-PCR with primers IK01+IK03 (iTaq, 20 ml total volume): | * no positive PCR of colony yet: run colony-PCR with primers IK01+IK03 (iTaq, 20 ml total volume): | ||
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== 2013-07-23 == | == 2013-07-23 == | ||
- | [[File: | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-23.png|100px|thumb|right|Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) and grown in liquid culture with primers RB43+RB44. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR; lane 4: streaked BAP1 by Konrad, lane 5: BAP1-pMM64-pMM65 from 2013-06-11]] |
* run colony-PCR with primers RB43+RB44 (iTaq, 20 µl total volume, use 1 µl of liquid culture) | * run colony-PCR with primers RB43+RB44 (iTaq, 20 µl total volume, use 1 µl of liquid culture) | ||
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Latest revision as of 03:34, 5 October 2013
2013-07-22
- no positive PCR of colony yet: run colony-PCR with primers IK01+IK03 (iTaq, 20 ml total volume):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
12 | 95 | 60 |
68 ↓0.5°C | 30 | |
72 | 180 | |
23 | 95 | 60 |
62 | 30 | |
72 | 180 | |
1 | 72 | 600 |
1 | 12 | inf |
- primers RB43+RB44 (against sfp) iTaq, 20 µl total volume:
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
35 | 95 | 60 |
55 | 30 | |
72 | 180 | |
1 | 12 | inf |
- unspecific bands in colony, no sfp bands at all
- re-run sfp PCR (primers RB43+RB44), include streaked BAP1 by Konrad
- sfp present in BAP1, not present in BAP1-pLF03, colony
- makes no sense, Konrad streaked his cells from the same batch of competent cells
- transfer BAP1, BAP1-pLF03, colony to liquid culture LB, grow at 37°C
2013-07-23
- run colony-PCR with primers RB43+RB44 (iTaq, 20 µl total volume, use 1 µl of liquid culture)
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 300 |
35 | 95 | 60 |
55 | 30 | |
72 | 180 | |
1 | 72 | 600 |
1 | 12 | inf |
- no sfp present