16 replies to this topic

[pcrman]

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.

 

Thanx

Hi Rama,

 

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.

[et2b]

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.

 

best wishes