Team:Bielefeld-Germany/Labjournal/Molecular
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Revision as of 15:27, 31 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 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: |
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: |
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: |