Jie:LabNotes/CpgSeq/2009-6-22

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Revision as of 18:48, 23 June 2009 by >Sam Chiang
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Methyl_PCR for ASE validation

PCR on bisuphite conversed Jurkat-DNA

2x iQ supermix                         20             x   12          
Primer F (50uM)			0.5	        0.5 each
Primer R (50uM)			0.5             0.5 each
BS Jurkat gDNA (202ng/ul)     	        0.1	  	1.2
dH2O				         20		240
total				         40ul
The primers are 20061015_ASM_valid. I set the annealing temperature at 58C, 62C. 
96C 3min-> 40x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.

File:20090622 ASM 58C anneal.jpg20090622_ASM_58C anneal

File:20090622 ASM 62C anneal.jpg20090622_ASM_62C anneal

PCR on samples

 The following two genes demonstrate the ASM in all samples that SNPs were called. 
 LSP1: PGP1_F and IMR90_iPS
 MATN4: PGP1_L and PGP3_L
 The following two genes demonstrate the ASM in specific tissues:
 DNMT3B: PGP3L and IMR90_iPS
 MMP9: PGP1_L and PGP1_F
 The bisulfite comverted gDNA comes from:
 IMR09_iPS: 83.5ng/ul refer to LabNotes on [1]
 PGP1_F: 119.5ng/ul refer to LabNotes on [2]
 PGP1_L: 386.7ng/ul refer to LabNotes on [3]
 PGP3_L: 238.6ng/ul refer to LabNotes on [4]


2x iQ supermix                         20             x   4          
Primer F (50uM)			0.5	        0.5 each
Primer R (50uM)			0.5             0.5 each
BS Jurkat gDNA (202ng/ul)     	        0.1	  	0.4
dH2O				         20		80
total				         40ul
96C 3min-> 40x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.
58C for MATN4 and DNMT3B;
62C for LSP1 and MMP9.