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