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.

I am following your discussion here. I have very similar problem. My question is how do you describe your gel?! Was there a smear or nothing at all?

Hi, I am new to this forum and doing BS conversion with Epitech Bisulfite conversion kit. I am using 10ug DNA but i am getting only 50ng DNA after BS conversion. I am doing the quantification with Qubit from Invitrogen. Please suggest me to get high amount of DNA after conversion.

I think you cannot use too much starting DNA in order to get high DNA yield. What amount of DNA does the kit suggests you to use? Using too much starting DNA can lead to incomplete conversion of unmethylated cytosines to uracils. We don't usually measure DNA concentration after conversion, and to see amplification, we do two rounds of PCRs.

On the 260/280 topic. Bisulfite conversion does not only make your DNA single stranded, but also degrades the DNA. This results in many small fragments with an -OH group at the end, which will thus "pollute" your spectro profile in the range of the ethanol group, so that you 260/280 is no longer 'clean' .

@ people doing double stranded DNA quantification after bisulfite conversion: do a single stranded method and your yield will be better :-). Bisulfite conversion yields non-complementary DNA strands.