Team:Bielefeld-Germany/Labjournal/PCRProtocols

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PCR Protocols and Programs

Standard Protocols

[Standard Protocols] Standard protocols

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

Specific Protocols

[Cytochromes] 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:

[Cytochromes] 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:

[Cytochromes] 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:

[Cytochromes] Phusion 50ul for psB1C3 with and without Gibson-overlap for mtrCAB

Reagent	1x	8x
GC-Buffer 5x	10.0	88.0
dNTPs (10mM)	2.0	17.6
Primer1 (10mM)	1.25	11.0
Primer2 (10mM)	1.25	11.0
Template (5.5ng/ul)	1.0	8.8
DMSO	1.5	13.2
Phusion (0.5u)	0.5	4.4
H2O	32.5	286.0

Program Phusion
98 °C	98 °C	70 °C	72 °C	72 °C
45s	30s	1:00	45s	5:00
	35 cycles
Notes: Size: 2070bp Primer1_Gibson: Primer2_Gibson: Primer1: Primer2:

[Cytochromes] Phusion 50ul for mtrCAB cluster

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
30s	10s	30s	1:15	6:15
	35 cycles
Notes: Size: 5150bp Primer1: Primer2:

[Cytochromes] Phusion 50ul for ccmAH cluster

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 (5.5ng/ul)	3.0	26.4
DMSO	3.0	26.4
Phusion (0.5u)	1.0	8.8
H2O	29.5	255.2

Program Phusion
98 °C	98 °C	68 °C	72 °C	72 °C
30s	10s	30s	1:35	6:00
	35 cycles
Notes: Size: 6311bp Primer1: Primer2:

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