Jie:LabNotes/CpgSeq/2009-6-22: Difference between revisions

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[[Image:20090622_ASM_62C anneal.jpg]]20090622_ASM_62C anneal
[[Image:20090622_ASM_62C anneal.jpg]]20090622_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
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.

Revision as of 18:36, 23 June 2009

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
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.