Integrity of treated DNA - Is quality of bisulfite treated DNA poor? (Jul/30/2009 )
Hello Everybody,
I have a question.
I am able to recover very less DNA ( only 140ng) after I treat my genomic DNA ( about 300ng) with bisulfite ( EZ DNA methylation-Gold Kit). Also, the quality of DNA is not good as indicated by the Nanodrop spec ( 260/280 = 2 & 260/230 = 3).
It would be great if someone could tell me if this is normal. If so, is it Ok for use in PCR.
Thanks.
-methdetector-
methdetector on Jul 30 2009, 10:29 PM said:
Hello Everybody,
I have a question.
I am able to recover very less DNA ( only 140ng) after I treat my genomic DNA ( about 300ng) with bisulfite ( EZ DNA methylation-Gold Kit). Also, the quality of DNA is not good as indicated by the Nanodrop spec ( 260/280 = 2 & 260/230 = 3).
I have a question.
I am able to recover very less DNA ( only 140ng) after I treat my genomic DNA ( about 300ng) with bisulfite ( EZ DNA methylation-Gold Kit). Also, the quality of DNA is not good as indicated by the Nanodrop spec ( 260/280 = 2 & 260/230 = 3).
A 260/280 ratio of 1.9 to 2.1 is quite normal for a bisulfite-converted DNA. Recovery is indeed very low and might be caused by either an unefficient DNA-binding step or an incomplete elution. Try the following:
Add ethanol of an equal volume of the binding buffer to the binding buffer / DNA mixture and incubate at RT for 15 minutes. Maybe this requires two loading steps.
Pre-heat the elution buffer to 50�C before adding to the spin column. Extend the incubation time to approximately 5 minutes.
Hope that helps...
MoB
-MoB-