Sau3AI partial digestion problem!!!

Stephen Smith sgsmith at tcd.ie
Thu May 4 15:40:41 EST 2000


Rodney,
This is the protocol I previously used to partially digest bacterial
genomic DNA. I'm not sure how applicable it is to your own situation.

Mix 50-100 micrograms of DNA with 100 microliters of Sau3A reaction buffer,
bring the volume of the reaction up to 1000 microliters with water and mix.

Dispense 100 microliter aliqouts of the above mix into eppies marked 2 to 9
and 200 microliters into eppie 1 .
Add one unit of Sau3A to tube 1, mix and transfer 100 microliters of this
to tube 2, mix and transfer 100 microliters of this to tube 3, continue
doubling dilutions to tube 8. Keep tube 9 as a no enzyme control.
Incubate at 37oC for 30 mins.
Stop the reactions by adding EDTA to 50 mM.
Electrophorese *only* 20 microliters, (very visible on gel since theres
about 1 - 2 micrograms of DNA ). This leaves you with 80 microliters to
play around with once you've decided which reaction gives you the best
partials.

Hopefully, of some help!
Best regards
Stephen


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