Zymo EZ Methylation Gold DNA bisulphite treatment - No DNA present after treatment (Feb/28/2010 )

Hi

I have been investigating DNA methylation and am having trouble with the Zymo methylation gold kit. I used the kit with 500ng of DNA and performed PCR afterwards but obtained no bands.

Then I used both the optimal amount of input DNA (500ng) and the maximum amount of input DNA (2ug) for a methylated positive control and a non unmethylated negative control. I have followed the protocol exactly and eluted in 10uL. When I run the input and eluted product on a 2% agarose gel, there is DNA present in the positive input sample but only a band present in the positive control at 2ug. I don't understand where the rest of the DNA has gone.

I was wondering if anyone had used the kit and experienced any similar problems and whether people knew any modifications to the protocol to improve the DNA recovery.

Thanks

Do you think an agarose gel is a sensitive method to detect DNA? Just keep in mind that bisulfite-converted DNA is no longer double-stranded and that ethidium bromide does interact only with unconverted and therefor dsDNA (not very likely with the EZ Methylation Gold Kit) or with regions with extremely low content of cytosines which remains nearly double-stranded. I strongly recommend UV quantification after bisulfite-treatment.

Hope that helps.

MoB

go ahead with PCR.

I experienced the same problem and this is what the tech support at Zymo replay:

Gel electrophoresis is one way to examine bisulfite converted DNA, but it will be difficult to see. We recommend that you chill the gel at 4 C for 15 minutes prior to looking at it under a UV lamp. We don’t know why this makes a difference, but it does! Check pg. 41 of our technical newsletter (http://www.zymoresearch.com/zrc/pdf/peanuts6.pdf) for an image. Another reason it is difficult to see bisulfite converted DNA on a gel is that the conversion process does introduce double strand breaks, so you will have a smear of DNA, not a fine band. Since you started with 260 ng of DNA and ran 2 uL of the product on the gel, you are only examining a small fraction of the DNA, possibly too little to see. You may have to use a Nanodrop or other method of spectrophotometry to measure concentration. Finally, the best method to check if you have had proper bisulfite conversion is to perform PCR with primers that have been validated for bisulfite converted DNA.

I hope this help,
good luck!

Hi

Thanks everyone for their suggestions.

I have used a Nanodrop to try and quantify the bisulphite treated DNA but i didn't obtain any readable results in the dsDNA, ssDNA and RNA programs when the input DNA was 500ng. I have since run several PCR with these samples with bisulphite primers and methylation-sensitive primers and have obtained no bands at all.

Basically it seems as though the DNA is not present after the EZ methylation Gold treatment.

Any advice as what to do next would be greatly appreciated.

Thanks

Hi,


I also tried the Gold version, but also didnt got it to work on 0.5 ug. dont know why. But every talking at conferences I noticed that some labs cannot get kits to work, while another lab swears on their mama's honour that's it is the best thing around.

for low amounts of DNA try the Qiagen kit and use the formaldehyde version of the protocol. It works perfectly on even much smaller amounts and it is roughly the same price as the zymo gold.

Otherwise, use the normal zymo kit, we still use it to great satisfaction for 0.5 ug, using both the columns as the handy and nifty 96-well format.

Hope it helps,

best

DNAlouie on Mar 2 2010, 06:19 PM said: