Large Scale Plasmid DNA Restriction Endonuclease Digest

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Tue Feb 8 16:52:00 EST 1994


In article <8FEB94.15253115 at skyfox.usask.ca>
weston at skyfox.usask.ca (Brigitte Weston) writes:

> I have a large quantity of plasmid (pUC deriviative) DNA to digest
> with :
> 1) EcoRI
> 2) EcoRI and HindIII double digest.
> What is the largest amount of plasmid DNA that anyone has digested at any one
> time (one reaction tube)?

Megatons ;-) Really...I don't think it matters how much material you want
to digest as long as you have enough enzyme.

> I have approximately one milligram of purified
> plasmid DNA which needs to be restricted in as short a time as possible.

Normally 1 Unit of enzyme will digest 1 ug of DNA in 1 hour at 37 C, so
for 1 mg DNA = 1000 ug you'll have to add a minimum of 1000 units to digest
your sample in an hour. If you add more, you could get away with less time.
To do the double digest, you could use a buffer compatible with both EcoRI
and HindIII such as a medium salt buffer containing 100 mM NaCl and add both
enzymes to the reaction mix.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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