Team:Heidelberg/Templates/MM week9

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2013-06-24

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2-4: negative control (BAP1-pLF03); lanes 5-10: colony-PCR of colony electroporated with purified PCR product; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03 (positive control); lanes 4,7,10: primers IK05+IK06 (negative control)

no colonies on 10 µl plate, 2 colonies on rest plate
colony-PCR using primer pairs IK01+IK02, IK01+IK03 (positive control), IK05+IK06 (negative control) (Taq, 20 µl total volume)

Cycles	temperature [°C]	Time [s]
1	95	300
30	95	60
	62	30
	72	60
1	72	600
1	4	inf

inconclusive results: no band for IK05+IK06 in BAP1-pLF03 (negative control)
grow liquid cultures at 37°C

2013-06-25

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB ON. Lane 1: NEB 2-log; lanes 2-5: negative control (BAP1-pLF03); lanes 6-13: colony-PCR of colonies electroporated with purified PCR product; lanes 2,6,10: primers IK01+IK02; lanes 3,7,11: primers IK01+IK03 (positive control); lanes 4,8,12: primers IK05+IK06 (negative control); lanes 5,9,13: primers IK01+IK06 (negative control)

repeat PCR with 1 µl of ON cultures:

Cycles	temperature [°C]	Time [s]
1	95	300
12	95	60
	68 ↓0.5°C	30
	72	120 (IK01+IK02; IK01+IK03;IK05+IK06) / 240 (IK01+IK06)
18	95	60
	62	30
	72	120 (IK01+IK02; IK01+IK03;IK05+IK06) / 240 (IK01+IK06)
1	72	600
1	4	inf

genomic integration failed
pick colony from BAP1-pKD46 plate from 2013-06-04, grow in 3 ml LB+Amp at 30°C

2013-06-26

inoculate 50 ml LB + Amp + Ara(0.5%) with 1.1 ml of ON culture
grow at 30°C to OD=0.73, prepare electrocompetent BAP1-pKD46

2013-06-27

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: 1 µl of PCR amplificate (first PCR)

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: 1 µl of PCR amplificate (second PCR)

Gel electrophoresis of the PCR amplificate. Lane 1: 19 µl of amplificate from first PCR; lane 2: 19 µl of amplificate from second PCR; lane 3: NEB 2-log ladder; lane 4: 1 µl of PCR amplificate (third PCR)

run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers IK07 and IK08 using Phusion polymerase (20 µl total volume) on cycler 1 (does not cool down to 4°C) (using hot start):

Cycles	temperature [°C]	Time [s]
1	98	30
30	98	5
	66	30
	72	150
1	72	600
1	4	inf

no product -> repeat PCR with cycler 2 (fully functional)
no product -> run 2-step PCR with 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Phusion polymerase (20 µl total volume, using hot start) on cycler 2:

Cycles	temperature [°C]	Time [s]
1	98	30
30	98	5
	72	150
	1	72	600
1	4	inf

no product

2013-06-28

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: 1 µl of PCR amplificate

run PCR of 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Q5 polymerase (20 µl total volume, using hot start) on cycler 2:

Cycles	temperature [°C]	Time [s]
1	98	30
30	98	5
	66	30
	72	150
1	72	1800 (30 min)
1	4	inf

perfect PCR => Phusion is crappy
run 4 PCRs of 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Q5 polymerase (50 µl total volume, using hot start) on cycler 2:

Cycles	temperature [°C]	Time [s]
1	98	30
45	98	5
	66	30
	72	150
1	72	1800 (30 min)
1	4	inf

2013-06-29

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lanes 2-5: 1 µl of PCR amplificate

load 1 µl of PCR product on gel -> specific amplificate
pool products (also include amplificate from 2013-06-28), purify (only first step, as no DpnI available)

2013-06-30

inoculate 1 ml LB+Amp with 10 µl of ON culture from 2013-06-25, grow at 30°C