DNA yields after Bisulfite Treatment - How can I optimize when I have low yield? (Oct/28/2010 )

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Hi MoB,

I didn't use carrier RNA because I started with >100 ng DNA. I guess a way to find out at this point is to do PCR and see if it works or not..
Oh and by the way, when you nanodrop the converted DNA..did you get an accurate quantification? say, if you start your conversion with maybe 200 ng of DNA, did the nanodrop reading show that you end up with the same yield of around 200 ng too...

Thank you!!

FNAD

From my experiences it's very difficult to produce a 'bad converted' DNA, especially with bisulfite-kits like the EpiTect- or the Zymo-kit. Either the DNA is converted or it remains completely unconverted. For a partially converted DNA I had to dilute the bisulfite reagent at least by the factor 1:10. And even then 260/280 ratios were in the range of 1.9 to 2.1. I have never obtained ratios around 3.0 to 4.0. Do you use carrier (RNA, glycogen, BSA, poly(d)A)? Maybe this might have an impact on the UV measurement?

Try measuring your bisulfite converted DNA in a NaOH solution using a factor of 33 to calculate concentration.

Hello!

I'm having the same problem! I couldn't amplify any of my 5 pairs of primers.

I started with 200ng and after the bisulfite conversion I ended up with 40ng. I used Nanodrop to quantify my samples and the 260/280 ratio ranges from 2,65 to 3,17.
I'll try to convert the DNA again to see if it helps me. I used RNA as carrier, so I'll do it without the RNA this time.