Team:Heidelberg/Templates/MM week12
From 2013.igem.org
(Difference between revisions)
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== 2013-07-15 == | == 2013-07-15 == | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-15_1.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-15_1.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) and grown in liquid culture with Cm+IPTG(1mM) with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: 1 µl of liquid culture.]] |
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-15_2.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-15_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 IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: 1 µl of liquid culture.]] |
* BAP1 did not grow on Amp, BAP1-pKD46 did grow on Amp at 37°C => no discrimination between pLF03 and pKD46 possible | * BAP1 did not grow on Amp, BAP1-pKD46 did grow on Amp at 37°C => no discrimination between pLF03 and pKD46 possible | ||
* Taq might have problems amplifying 4.8 kb from genomic DNA => use Phusion Flash (primers IK07+IK08, 20 µl total volume, use 1 µl of liquid culture, old BioRad cycler): | * Taq might have problems amplifying 4.8 kb from genomic DNA => use Phusion Flash (primers IK07+IK08, 20 µl total volume, use 1 µl of liquid culture, old BioRad cycler): | ||
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== 2013-07-18 == | == 2013-07-18 == | ||
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-18_1.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-18_1.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 2,4,6: BAP1-pLF03 (control); lanes 3,5,7: colony-PCR. Lanes 2,3: primers IK24+IK25; lanes 4,5: primers IK07+IK25; lanes 6,7: primers IK01+IK25]] |
- | [[File:Heidelberg_BAP1-colony-PCR2013-07-18_2.png|100px|thumb | + | [[File:Heidelberg_BAP1-colony-PCR2013-07-18_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). Lane 1: NEB 2-log; lanes 2,4,6: BAP1-pLF03 (control); lanes 3,5,7: colony-PCR. Lanes 2,3: primers IK24+IK25; lanes 4,5: primers IK07+IK25; lanes 6,7: primers IK01+IK25]] |
* run colony-PCR with primers IK24, IK25 (OneTaq, 20 µl total volume): | * run colony-PCR with primers IK24, IK25 (OneTaq, 20 µl total volume): | ||
{| class="wikitable" | {| class="wikitable" |
Latest revision as of 02:31, 5 October 2013
2013-07-15
- BAP1 did not grow on Amp, BAP1-pKD46 did grow on Amp at 37°C => no discrimination between pLF03 and pKD46 possible
- Taq might have problems amplifying 4.8 kb from genomic DNA => use Phusion Flash (primers IK07+IK08, 20 µl total volume, use 1 µl of liquid culture, old BioRad cycler):
Cycles | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
35 | 98 | 1 |
66 | 5 | |
72 | 100 | |
1 | 72 | 600 |
1 | 12 | inf |
- control did not work -> repeat in new BioRad cycler (pick from plate)
- same result
2013-07-18
- run colony-PCR with primers IK24, IK25 (OneTaq, 20 µl 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 |
- 1.2 kb band with primers IK01+IK25 in control => makes no sense, repeat
- same result
- transfer BAP1-pLF03 to new Cm+IPTG plate, colony 2 to LB w/o antibiotics, grow at 37°c#