Team:Bielefeld-Germany/Labjournal/PCRProtocols

From 2013.igem.org

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<h1>PCR Protocols and Programs</h1>
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<h1 style="color:#ff6600;">PCR Protocols and Programs</h1>
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<h2>Standard Protocols</h2>
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<h2>Specific Protocols</h2>
<h2>Specific Protocols</h2>
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Latest revision as of 11:35, 26 September 2013







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 °C98 °C68 °C72 °C72 °C
30s 10s30s 1:356:00
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 °C98 °C60 °C72 °C72 °C
1:00 10s30s 30s6:00
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 °C98 °C70 °C72 °C72 °C
30s 10s30s 50s6:00
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 °C98 °C70 °C72 °C72 °C
45s 30s1:00 45s5:00
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 °C98 °C60 °C72 °C72 °C
30s 10s30s 1:156:15
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 °C98 °C68 °C72 °C72 °C
30s 10s30s 1:356:00
35 cycles
Notes:
Size: 6311bp
Primer1:
Primer2:







































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