IUBio

Free molecular biology program.

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Thu Mar 18 23:22:01 EST 1999


> colinw wrote:
> 
> > If you work with restriction endonucleases alot, you might find this
> > little
> > program I wrote useful.
> > It integrates enzyme/buffer activity information from several different
> > restriction endonuclease manufacturers into a database.  With
> > it, you can select the enzymes you want to use in a multiple restriction
> > enzyme digest and have the program give you a list of buffers that will
> > work for all of them (saves a little time looking through catalogs and
> > activity tables).
[snip]
> >              http://users.mis.net/~colinw/buffer.html
> > colin wilson

I tried it.  I like it.  I found two "gotchas"

a) Star activity

For example your program claims that EcoRI is 100% active
in most buffers.  This is true but it is not specific in
most of them but will display star activity.  If you check
eg. the double digest table in the EcoRI catalogue you will
find that the special "EcoRI" buffer is recommended for all
digests, and other companies suggest their own buffer with
the highest salt concentration.
     To remedy this you will have to read the descriptions
of each of the enzymes and flag any of those that can do
this.  The same would hold in situations where star activity
is triggered by excess glycerol or enzyme.

b) Temperature

The activity lists in the catalogues tend to use a sliding
scale with 100% or +++ or whatever being the best activity.
This "best" may not be 100% of the actual (enzyme Unit)
value if you incubate at a non-optimal temperature.  For example
your program predicts that both SphI and BstNI would have 100%
activity in buffer NEB2.  In this case "100%" for BstNI would
be 30% of its true (Unit) activity as this enzyme must be
incubated at 60degC for full activity.
     Again, you can remedy this by flagging any enzyme with
a strange temperature requirement.  While your eventual
choice of buffer would be correct you would have to add the
enzymes sequentially after the switch in temperature.

While your program certainly helps cut down the number of
choices there's still no substitute for knowing the habits
and preferences of the enzymes in your freezer (eg. stability
in a reaction, ease of inactivation, sensitivity to methylation,
ease of cutting at DNA ends, cost etc.).
     Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA




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