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.