Large Scale Plasmid DNA Restriction Endonuclease Digest
wes...@skyfox.usask.ca
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)? I have approximately one milligram of purified
plasmid DNA which needs to be restricted in as short a time as possible.
Thanks in advance.
Brigitte Weston.
Song Tan
I've digested 2 mg of a pUC based plasmid with 1000 units of EcoRI and 1000
units of PstI in 1 ml final volume (overnight at 37C, regular Eppendorf tube).
I didn't try to optimize the amounts of EcoRI or PstI (both are reasonably
inexpensive). Note that the plasmid contained only one EcoRI and one PstI
site. I think that your EcoRI single digest should go without any problems.
So should the EcoRI + HindIII double digest, although if you have problems with
the HindIII digesting very concentrated DNA samples, you might try lowering the
DNA concentration. Thomas Rechsteiner in our lab has found that lower substate
concentration can improve HindIII digestion (i.e. less HindIII needed).
As to your last question, we digest 50 - 200 mg of plasmid in one reaction
vessel several times a year (!).
Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: t...@aeolus.vmsmail.ethz.ch
Paul N Hengen
wes...@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: p...@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 /--------------------------/*
*******************************************************************************
Peter M. Muriana
..........stuff deleted........
>As to your last question, we digest 50 - 200 mg of plasmid in one reaction
>vessel several times a year (!).
>
>Song Tan
>Institute for Molecular Biology and Biophysics
>ETH-Honggerberg (Swiss Federal Institute of Technology)
>8093 Zurich, Switzerland
Just out of curiosity, what do you do with such large amounts of digested
vector/plasmid DNA? - saving it for future use or selling it as a digested
vector? You mentioned **mg** and not **ug**, right?
ne...@mbcf.stjude.org
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: ne...@mbcf.stj.org
St. Jude Children's Research Hospital Phone: (901) 522-0400
Memphis, TN Fax: (901) 523-2622
Song Tan
Muriana) writes:
>In article <2j8jfu$r...@elna.ethz.ch>, t...@aeolus.vmsmail.ethz.ch (Song Tan) writes:
> ..........stuff deleted........
>>As to your last question, we digest 50 - 200 mg of plasmid in one reaction
>>vessel several times a year (!).
>
>vector/plasmid DNA? - saving it for future use or selling it as a digested
>vector? You mentioned **mg** and not **ug**, right?
>
Biophysical studies is the short answer.
I know that digesting multiple mg of plasmid sounds like an inordinate amount,
but it's not when you're trying to isolate insert DNA to be used for
biophysical studies such as crystallography (see for example Richmond et al.,
Crystals of a nucleosome core particle containing defined sequence DNA, JMB,
199, 161-170, 1988).
I found a paper from 1981 which mentions preparing 350 to 1600 mg of plasmid
from 1000 g of packed cells (300 liter fermenter run), and digesting 250 mg of
plasmid with EcoRI ! (Hillen et al., Preparation of milligram amounts of 21
deoxyribonucleic acid restriction fragments, Biochemistry, 20, 3748-3756,
1981). And I'll bet there are other examples in the literature.
An aside: the Hillen paper mentions a nice way of fractionating DNA fragments
by PEG precipitation, even if the fragments have sticky ends. PEG
precipitation will fractionate blunt-ended DNA fragments by size, but doesn't
work so well with sticky-ended fragments presumably because of interactions of
the sticky ends. Addition of gelatin to 0.3% allowed fractionation of the
sticky-ended EcoRI fragments by PEG precipitation.
Song Tan
(Tim Richmond lab)
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: t...@aeolus.vmsmail.ethz.ch