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Digestion time and enzyme


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#1 netnus

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Posted 05 October 2004 - 04:19 PM

My friend told me today that it wasn't good to digest the PCR insert with the restriction endonucleases for very long time. It would cast a bad influence on the following ligation. Is it right? Why?

After digestion, Do you inactivate the enzyme by heating up the mixture?I digest my insert with BamH. Unfortunately Bamh can't be killed by heating.
Anyone could give me some suggestions except gel purification that I could remove the BamH from the digested mixture ?


thanks!

netnus

#2 kant0008

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Posted 05 October 2004 - 09:24 PM

Hi,
as for time- how long is too long? Some enzymes have "star" activity, they start cutting non-specifically if at too high concentration or if left too long, I'm not sure if Bam HI has it though. I don't think there are any specific requirements for cutting PCR products.
To get rid of your enzyme you can ethanol precipitate your mixture.




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