Kun Zhang LabNote Oct week3
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Sunday 2007-10-14[edit]
Sun.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
Monday 2007-10-15[edit]
Mon.Exp1: Fixing the PGP1Ver2 PCR reactions I did on 2007-10-12[edit]
- Since I haven't run the thermalcycling on the 96 reactions on cDNAs, I'm just adding the ThermoStart Taq enzyme and more ABI's dNTPs into the reactions.
10x buffer 0.1 x100 10 ThermoStart Taq 0.25 x100 25 10mM dNTP 0.5 x100 50 H2O 0.15 x100 15
- Also set up 32 reactions on the genomic DNA template.
Reaction set up:
' | one reaction | 33 reactions |
Template | 1 | each |
10x Buffer | 5 | 165 |
25mM MgCl2 | 3 | 100 |
10mM dNTP | 1 | 33 |
10uM primer mix | 1 | each |
50x SYBG I | 0.4 | 13.2 |
ThermoStart Taq | 0.25 | 8.25 |
H2O | 38.35 | 1267 |