nested deletions

[Gary K.]

In article <412ds2$42t at lyra.csx.cam.ac.uk>, rw200 at cus.cam.ac.uk (R.
Woodward) wrote:

> Hello everyone
>  Ineed a bit of advice re: nested deletions. I have been using a kit
supplied by NEB ('Exo Size deletion kit') the problem is that the only
plasmid I can rescue are of parental size evne though I can see that the
ExoIII has worked very nicely from analytical gels of samples taken at
different time points of my deletion reactions. I suspect the Mung Bean
Nuclease is to blame (the reaction vol is 20ul for 30min at 30oC using
50mMNaOAc pH 5.0, 30mM NaCl and 1mM ZnSO4). Is there anything I should be
aware of a
> bout MBN any suggestions etc.... I have used the Pharmacia deletion kit
in the past and that worked o.k. but that used S1 nuclease. Whats the
feeling should I buy some S1 nuclease to use in my present kit?  Any other
thoughts would be welcomed. Thanks v.much to all of those poeple who have
answered my previons questions.
>  Robert
> 
>  R.Woodward
>  Email rw200 at cus.cam.ac.uk
>  Dept Pharmacology
>  Tennis Court Rd
>  University of Cambridge
>  U.K.
> vi -r /usr/tmp/nn.a04526



I have had good success making nested deletions using the following 5X
Mung Bean Nuclease buffer:

   150mM NaOAc pH 5
   250mM NaCl
   5mM ZnCl2
   25% Glycerol

I incubate either at 30 C for 30 min, or 37 C for 10 min.

Good luck,
Gary