Re: How to Perform a Partial Digestion!

["Murray, Elizabeth" <murraye@MARSHALL.EDU>]

Hi!
I used to work for Promega doing RE enzyme testing.  Promega did overpack
enzymes a little to prevent them dying in your freezers (as do many others)
but only by 2-4 units.  Your label might say 10 units and we would give you
12-14.  They could lose money otherwise and too much enzyme tends to leave
star activity.  One possibility is to trick the enzyme by using the wrong
buffer.  In the Promega catalog (or their RE guide on line) you will notice
some buffers (I think A,B,C, D and H are listed) cut 25% or 50% vs. 100% for
the recommended buffer.

http://www.promega.com/reguide/chapthree/table3_1.htm

This can give you some good advice for reducing the efficiency of your enzymes.
The tricky thing is that some enzymes like Hind III will keep their activity
as you dilute them out.  Some enzymes have preferred sites that will cut
more quickly, making a partial at those sites problematic if you just
shorten the time.  Some enzymes will cut to completion in 5 minutes on a
nice clean plasmid  because their unit definition was defined on a substrate
like Lambda with 5-6 sites, not 1 or 2 sites.  Some enzymes (like Sal I)
actually don't like to cut circular plasmid.  I think altering the salt in
the buffer could be your best bet, plus try a less than 1 unit per ug DNA
(between 1/2 unit/ug and 1/10) for an hour.  Also, try leaving the BSA out
of the RE digest if you are used to adding it, as that only enhances
cutting.  Genomic DNA is a lot easier to do a partial on as it tends to be
sort of dirty.
I also recommend you check out Promega's technical service if it is an
enzyme that is sold by them- they have lots of useful information on their
enzymes since they actually manufacture most of them themselves and they are
very responsive.  NEB may have a similar data table for some of their
buffers and enzymes, as they also manufacture most of them themselves and
they are also helpful.
Good luck,
Liz Murray

 -----Original Message-----
From:   Mahmoud Yaish [mailto:degmya@UNILEON.ES]
Sent:   Tuesday, February 20, 2001 8:19 AM
To:     plant-tc@tc.umn.edu
Subject:        Re: How to Perform a Partial Digestion!

> Dear Friends,
>
> I am doing plasmid construction and will use partial digestion for it. But I
> don't know how long is good for partial digestion for a standard reaction. I
> really appreciate your suggestions.
>
> Yours sincerely,
>
> Daolin Fu
> Kansas State University
>
> Tel: 785 532 7226
> Email: dlfu@ksu.edu
>
Dear Daolin,
I think you can do partial digestion using at least two of the following
methods:
Check the minimum time that is necessary to realize your fragment during the
digestion or using ethidium bromide at several concentrations in your digestion
procedure. 
Sincerely Yours,

Mahmoud

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