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How to detect incomplete enzyme digests


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#1 mjcramer

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Posted 05 February 2009 - 02:43 PM

I'm performing enzyme digestions to detect methylation of the genome at certain locations and am worried that I'm getting an incomplete digest. I'm wondering what are some methods for detecting incomplete digestions?

Thank you,

mjcramer

#2 pcrboy

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Posted 06 February 2009 - 12:13 PM

controls, controls, controls.

#3 perneseblue

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Posted 08 February 2009 - 03:54 AM

well how about cutting samples of your DNA for progressively longer periods. 1hr, 2hr, ... overnight.
May your PCR products be long, your protocols short and your boss on holiday

#4 klinmed

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Posted 08 February 2009 - 04:40 AM

Would suggest you perform your digest as usual. Take sample after incubation and analyse (gel? or whatever). Then to the bulk reaction mix add a further aliquot of enzyme, incubate and reanalyse. If you get the same result from both reactions then primary digestion should be ok.

If you are worried about the presence of inhibitors in your sample, it can be useful to compare runs with different amounts of starting sample ( if your analysis method permits.

Hope this helps.




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