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restriction reaction - (Jun/28/2002 )

how can one decide the amount of DNA to be digested?I mean, when we degist 20ng or 50ng of DNA in one digestion reaction,where is the 20ng or 50ng come from?


the protocol tell us there should be 20ng or 50ng of DNA in one digestion reaction,but reality is not equal the fact, we needn't count the amount of the DNA. if you can see a bright band in a agarose electrophoresis by 3ul DNA, the digestion of 10ul DNA can be well done. such as production of PCR, or comercial kits of plasmis extraction.  


read the unit definition fro the enzymes you are using.  Boehringer enzymes have the following unit definition.  "one unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1 µg substrate DNA (or fragments) in 60 minutes at the appropriate temperature under optimal assay conditions as stated for each restriction endonuclease."  This also depends upon the number of restriction sites in the substrate DNA however.  I was reliably informed that Lambda DNA was the standard substrate used for this purpose.  Hope that helped.