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[methdetector]

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.

[MoB]

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).

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