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.