Digestion time and enzyme - (Oct/05/2004 )
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
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.
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.
You can use the phenol purification step or Micropure Enzyme Removal comlumns, not so sure if they work on your enzyme though. As for star activity, it was reported for BamHI and it can be due to overdigestion, meaning if you leave the reaction to go on too long as you said, but you can always go by the definitkion of enzyme units and then predict how long your reaction should be.