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5-aza-2'-deoxycytidine inhibits digest?


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#1 Substance

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Posted 19 July 2009 - 03:54 AM

So I'm doing a CpG island methylation assay where i have 4PCRs with RE-sites between the primers.

The idea is to digest away ummethylated DNA and leave methylated DNA as template for a PCR.

I'm trying to digest gDNA with a bunch of different methylation sensitive enzymes (AciI,BssHII,NotI-HF,NaeI,HpyCH4IV).

Im testing out the system on three different DNAs+ctrl;

1)NEB Jurkat CpG-methylated DNA
2)NEB Jurkat 5-aza-2'-deoxycytidine (mostly) unmethylated DNA
3)Cancer cell line DNA
4)A PCR product as a digest control

After over-night digestion (+/- enzyme) i run PCRs on ~5ng of digested DNA and the 5-Aza thing outperforms the CpG methylated DNA every single time, even though it should be mostly unmethylated (-->cleaved).

Questions:
-Since I'm going to be using primary tumor DNA later it's important to be able to use as little input material as possible, is 10-20ng too little in a 10 or 20ul digest?
-If 10-20ng gDNA is too little, can I spike my digest mix with e.g. a random PCR product (to increase the DNA conc without adding any template for the subsequent PCR), or is that just stupid?
-Can 5-aza-2'-deoxycytidine inhibit RE-digest (since the PCRs dont show much of a difference between +/- enzyme as template)?

Note; the digest is done according to manufacturers protocol (+BSA), for 16h in a volume of 10 or 20 ul, the control PCR product (1ug) is completely cleaved.

#2 Substance

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Posted 21 July 2009 - 08:20 AM

So no one in this entrie forum has any thoughts on my problem?

I must say I'm a bit dissaponted, but Ill bump this up once and hope for an answer (Ill take speculations, guesses, hail marys, anything).




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