hi,
I am trying to pull down methyl rich genomic regions using MedIP, but my negetive control ( igG ) shows equivalent enrichment as test.
I isolate genomic dna, sonicate it using diagenode sonicator, give denaturation for 10 mins at thermocycler PCR machine followed by 10 mins incubation at ice. Add MedIP buffer, reserve 25 ul for input and add antibody; give overnight 4 degree incubation on tube rocker
post incubation I add beads and incubate at 4 degree on rocker for 1 hr. and the elute in elution buffer with proteinase k (20 ug) at 55 degree, 3 hrs.
upon performing qPCR the cp values for test(5mc antibody ) and negetive control are same. I am stuck at this problem since months if anyone could please suggest me with something???????
Your protocol seems fine to me. Are you using some kit? One suggestion is change your normal IgG. And you can increase the number of wash steps also (I usually wash 4 times after IP and performed fifth wash with high salt wash buffer).
Best
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