Kun:LabNotes/ASE/2007-10-14
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Exp1: PCR debugging[edit]
In this experiment, I'm setting up a number of PCR reactions with different template DNA, batches of primers, dNTPs and Taqs. The purpose is to find out what went wrong in the last three experiments.
iTaq reactions
iTaq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Template, gDNA (10ng/ul) | HDAA50, 1ul | GM20431, 1ul | GM20431,cDNA, 1ul | HDAA50, 1ul | HDAA50, 1ul | GM20431, 1ul | GM20431,cDNA, 1ul | HDAA50, 1ul |
10X buffer | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul |
50mM MgCl2 | 1.5ul | 1.5ul | 1.5ul | 1.5ul | 1.5ul | 1.5ul | 1.5ul | 1.5ul |
10mM dNTP | ABI, 1ul | ABI, 1ul | ABI, 1ul | ABgene, 1ul | ABI, 1ul | ABI, 1ul | ABI, 1ul | ABgene, 1ul |
50X SYBG I | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul |
iTaq | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul |
H2O | 40ul | 40ul | 40ul | 40ul | 40ul | 40ul | 40ul | 40ul |
10uM Primer | rs1264899, 1ul | rs1264899, 1ul | rs1264899, 1ul | rs1264899, 1ul | rs1045216, 1ul | rs1045216, 1ul | rs1045216, 1ul | rs1045216, 1ul |
Thermo Start Taq
Thermo Start Taq | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Template, gDNA (10ng/ul) | HDAA50, 1ul | GM20431, 1ul | GM20431,cDNA, 1ul | HDAA50, 1ul | HDAA50, 1ul | GM20431, 1ul | GM20431,cDNA, 1ul | HDAA50, 1ul |
10X buffer | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul |
25mM MgCl2 | 3ul | 3ul | 3ul | 3ul | 3ul | 3ul | 3ul | 3ul |
10mM dNTP | ABI, 1ul | ABI, 1ul | ABI, 1ul | ABgene, 1ul | ABI, 1ul | ABI, 1ul | ABI, 1ul | ABgene, 1ul |
50X SYBG I | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul | 0.4ul |
Thermo Start Taq | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul |
H2O | 38.5ul | 38.5ul | 38.5ul | 38.5ul | 38.5ul | 38.5ul | 38.5ul | 38.5ul |
10uM Primer | rs1264899, 1ul | rs1264899, 1ul | rs1264899, 1ul | rs1264899, 1ul | rs1045216, 1ul | rs1045216, 1ul | rs1045216, 1ul | rs1045216, 1ul |
Thermocycling protocol:
95C 15min (3min for iTaq reactions) -> 40 cycles of (94C 30S -> 56C 30S -> 72C 1min) -> 72C 3min