Team:Bielefeld-Germany/Labjournal/Molecular

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Revision as of 20:27, 30 August 2013

PCR Protocols and Programs

Phusion 50ul for mtrCAB Fragment1

Reagent	1x	8x
Hf-Buffer 5x	10.0	88.0
dNTPs (10mM)	2.0	17.6
Primer1 (10mM)	1.0	8,8
Primer2 (10mM)	1.0	8,8
Template (16.5ng)	3.0	26.4
DMSO	4.0	35.2
Phusion (0.5u)	1.0	8.8
H2O	26.0	228.8

Program Phusion
98 °C	98 °C	68 °C	72 °C	72 °C
30s	10s	30s	1:35	6:00
	35 cycles
Notes: Size: 1800bp Primer1: Primer2:

Phusion 50ul for mtrCAB Fragment2

Reagent	1x	8x
Hf-Buffer 5x	10.0	88.0
dNTPs (10mM)	2.5	22.0
Primer1 (10mM)	1.0	8,8
Primer2 (10mM)	1.0	8,8
Template (5.5ng/ul)	2.0	17.6
DMSO	3.0	26.4
Phusion (0.5u)	1.0	8.8
H2O	29.5	259.6

Program Phusion
98 °C	98 °C	60 °C	72 °C	72 °C
1:00	10s	30s	30s	6:00
	35 cycles
Notes: Size: 330bp Primer1: Primer2:

Phusion 50ul for mtrCAB Fragment3

Reagent	1x	8x
Hf-Buffer 5x	10.0	88.0
dNTPs (10mM)	2.5	22.0
Primer1 (10mM)	1.0	8,8
Primer2 (10mM)	1.0	8,8
Template (5.5ng/ul)	2.0	17.6
DMSO	3.0	26.4
Phusion (0.5u)	1.0	8.8
H2O	29.5	259.6

Program Phusion
98 °C	98 °C	70 °C	72 °C	72 °C
30s	10s	30s	50s	6:00
	35 cycles
Notes: Size: 3000bp Primer1: Primer2:

Phusion 50ul for psB1C3 with Gibson-overlap for mtrCAB

Reagent	1x	8x
Hf-Buffer 5x	10.0	88.0
dNTPs (10mM)	2.5	22.0
Primer1 (10mM)	1.0	8,8
Primer2 (10mM)	1.0	8,8
Template (5.5ng/ul)	2.0	17.6
DMSO	3.0	26.4
Phusion (0.5u)	1.0	8.8
H2O	29.5	259.6

Program Phusion
98 °C	98 °C	70 °C	72 °C	72 °C
30s	10s	30s	50s	6:00
	35 cycles
Notes: Size: 3000bp -> 17sec/kb Primer1: Primer2:

Taq 25 µl

Reagenz	1x	8x
Buffer 5x	5,0	44
MgSO4	0	0
dNTPs	2,5	22
Primer1	1,0	8,8
Primer2	1,0	8,8
Template	1,5	13,2
DMSO	2,0	17,6
Polymerase	0,5	4,4
H2O	11,5	101,2

Program Taq
98 °C	98 °C	x °C	72 °C	72 °C
2 min	30 s	30 s	y	6 min
x: according to Primer Tm y: 15s/kb

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