5ug DNA can be converted in one bisulfite treatment - (Sep/03/2006 )

generally, we can only convert 2ug DNA in 1 bisulfite treatment. because i have lots of sample and primer to examine, i try to convert 5ug DNA in 1 treatment, after that, i check by routine PCR or COBRA.

i found, the conversion is complete, because i did not see amplification in routine PCR(EGFR primer), that means, no non-converted DNA remain.

i then dilute the the treated DNA in 2.5 times lowTE, and COBRA work well, so i guess i got 2.5 times more converted DNA.

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Hi gaodaxia,

thanks for sharing your experience.

What method or kit do you use for the conversion? Have you scaled up the reaction volume based on the DNA amount? What are your routine EGFR primers (any C's and CpGs in the primer)? Please remember incomplete conversion usually occurs in highly CpG rich areas, the center of a CpG island for instance.

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This has been my experience with time but not amount. I use the MethylEasy kit and usually convert 2ug at a time. On a couple of occasions I've converted 4ug (the maximium suggested) with good results.

I usually treat 2ug overnight (16hr) and this has always resulted in close enough to 100% conversion for me (this will yield 100ul of 20ng/ul converted DNA). I recently tried a 4 hr conversion (the minimum time recommended) and the non-CpG C conversion rate dropped to as low as 93 or 94% for some clones sequenced. The worst affected areas were those extremely rich in CpGs. For the sake of one night I wouldn't recommend a 4hr conversion with this kit. However, overnight conversion with up to 4ug DNA appears to work well, at least for the CpG islands I've been looking at.

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one of the things to be sure of is that ssDNA is required for efficent bisulfite conversion, there are steps within the method that use NaOH and incubation at 37C for denaturing the DNA. This is fine for single copy genes and loci, for repetitive sequences I find an additional step of 100C for five minutes immediately prior to addition of bisulfite helps with full conversion by bisulfite.

I think having so much DNA would not be an issue for methylation studies however having less (less than 100ng of starting material) would be more challenging, how low have people gone in terms of amount of starting genomic DNA/??

Nick

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I have had no problem converting 100ng with a kit but I have seen papers where the authors have prepared a lysate from no more than a 1000 cells and successfully used this for bisulphite conversion (I think some of the embryology papers go even lower).

The MethyEasy kit suggests 100pg conversions are successful but I've never tried going this low.

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Hello

Good morning. I've got a little problem. I'm studying methylation status with a kit named: Cpgenome DNA modification kit.
I started with an amount of 3µg of DNA and after using the kit i've got no DNA in my tubes. I don't know why can you help me?

Ilofleur.

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IloFluer,

how have you determined you have no DNA in your tubes?

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Hello methylnick!

I determined the dna amount by spectrophotometry. Is it bad to do that?

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Hi Ilofleur,

You can't determine amount of DNA by spectrophotometry after bisulfite modification as it s singlestranded and contains Uracil. The only way of knowing if the modification was successful is to have a product on the gel after PCR. You should use primers and conditions known to work from a paper or you can also buy special primers (the methyleasy Kit comes along with some).
So maybe you have lots of modified DNA in your tube ad your Kit does actually work

Good luck,

Krümel

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