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Integrity of treated DNA


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#1 methdetector

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Posted 30 July 2009 - 12:29 PM

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. :lol:

#2 MoB

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Posted 31 July 2009 - 12:18 AM

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 50C before adding to the spin column. Extend the incubation time to approximately 5 minutes.

Hope that helps...

MoB




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