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Genomic DNA Complete Digestion


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4 replies to this topic

#1 Samantha

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Posted 24 January 2005 - 08:36 PM

i digested my genomic dna (10ug) with a restrcition enzyme (100u) twice overnight, together with 0.1mg/ml BSA in a 50ul reaction. however, the digestion is not complete. the OD260/OD280 ratio is 2.

what can i do to make the digestion complete? what do you guys usally do for complete digestion of genomic dna ?

thanks

samantha

#2 pcrman

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Posted 24 January 2005 - 09:48 PM

I am curious how you know the digestion is not complete?

#3 Samantha

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Posted 24 January 2005 - 10:53 PM

i know the digestion was incomplete because I also set up a negative control. There was a very sharp thick band with a large size...which also appeared in the negative control. If the digestion was complete, the thick sharp band shouldnt appear and a smear should be seen instead. Actually, my genomic DNA was digsetd with HpaII which cleaves unmethylated CpG sites, so a smear was expected.

#4 peixumol

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Posted 25 January 2005 - 03:21 AM

try to further purify your DNA,because very few pollutants may inhibit the activity of the restriction enzyme.
paul in NanJing China
God helps them who help themselves!

#5 chandima

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Posted 25 January 2005 - 03:41 AM

hi
purity is pretty important, do organic extractions to the sample or u can purify it with commmertially available colums. i usually do twise, the organic extraction during DNA extraction.
chandima




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