Large Scale Plasmid DNA Restriction Endonuclease Digest

neale at mbcf.stjude.org neale at mbcf.stjude.org
Wed Feb 9 23:02:00 EST 1994


In regards to some of the suggestions being offered, I think two things should
be remembered about restriction enzyme units:

(1) The units quoted for the enzymes are usually based on linear DNA ie lambda

(2) The concentration of DNA in the digest is usually 1 ug per 50 ul

(and, of course, the units usually refer to the DNA being cut to completion in
one hour)


With respect to (1), NE Biolabs has a rough conversion chart for the relative
amount of enzyme required for circular plasmids vs a linear template. In most
cases the number of units for complete digestion is MORE than that required for
a linear template.

With respect to (2), 20 ug/ml is a very low concentration of DNA, but it is the
standard that the manufacturers rate their enzymes. In practice we try to limit
the DNA concentration to <250 ug/ml to maximize completeness of digestion. This
rule of thumb works well with all types of DNA in our hands, but we have
exceeded this on occasion with purified plasmid DNA but not past 1 mg/ml.

I hope this is helpful.


Geoff Neale

Dept. of Virology and Molecular Biology          Internet: neale at mbcf.stj.org
St. Jude Children's Research Hospital               Phone: (901) 522-0400
Memphis, TN                                           Fax: (901) 523-2622






In article <CKxE2p.Jop at ncifcrf.gov>, pnh at fcsparc6.ncifcrf.gov (Paul N Hengen) writes:
> 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.
> 
> *******************************************************************************
> * Paul N. Hengen, Ph.D.                           /--------------------------/*
> * National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
> * Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
> * Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
> * Frederick, Maryland 21702-1201 USA              /--------------------------/*
> *******************************************************************************



More information about the Methods mailing list