Jie:LabNotes/CpgSeq/2009-8-26
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ASM validation of cell type and individual dependent ASM[edit]
primers for cell and individual dependent ASM validation
I did the PCR using 2x iQ supermix on: rs10846023_FLJ22662 on BJ(7/26/09) and BJiPS11(; MATN4 on PGP1F(10/10/08) and PGP1iPS; DNMT3B on PGP3L and PGP9L; x 7 2x iQ supermix 50 350 Primer F (50uM) 0.5 0.5 each Primer R (50uM) 0.5 0.5 each sample bis-gDNA (~202ng/ul) 0.1 0.1 each dH2O 50 350 total 40ul
96C 3min-> 45x (95C 30sec, MATN4 and DNMT3B at 58C, rs10846023 on 62C 1min, 72C 1min)-> 72C 5min. File:20090826 MATN4 DNMT3B rs10846023 ASM.jpg20090826_MATN4_DNMT3B_rs10846023_ASM
TA cloning of the above 6 samples[edit]
Step 1. Cloning into pCR2.1.
Set up the 10ul ligation reaction as follows: x 6 DNA template 0.5ul 3 10x Ligation Buffer 0.5ul 3 pCR2.1 vector(25ng/ul) 1ul 6 ATP 10mM 0.5ul 3 water 2ul 12 T4 DNA ligase(4 Weiss Units)0.5ul 3 Incubate the ligation reaction at 14C overnight.