Team:Heidelberg/Templates/MM week12

From 2013.igem.org

2013-07-15

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.

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
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

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):

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#