Jie:LabNotes/CpgSeq/2009-6-22
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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 8 Primer F (50uM) 0.5 0.5 each Primer R (50uM) 0.5 0.5 each BS Jurkat gDNA (202ng/ul) 0.1 0.8 dH2O 20 160 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.