Q: How doing partial RE digests?

Marieke R. Koedood Zhao rkoedood at bio.bu.edu
Mon Jan 27 13:05:50 EST 1997


Definitely use the enzyme dilution scheme. In our lab everybody who tried
varying time repeated the experiment numerous time till switching to
varying enzyme dilution which worked every time.

Good luck.

Marieke


: Yes, you could try varying time. I prefer to keep this variable constant and
: modify the amount of enzyme I add, using serial dilution of the reaction into
: a fixed concentration of DNA. A reasonable overview of this technique is in the
: "Red Book". I summarize a reaction I did last week here:

:          XbaI/---------/BsrGI(undesired)/---------------/BsrGI(desired)

: After fully digesting ~2ug with XbaI in 50ul (50mM NaCl), I aliquoted the
: reaction into: 1x15ul,3x10ul,1x5ul. Put all on ice and added 2u BsrGI to
: 15ul tube. Mixed and serially diluted (4x(1:3)). Put at 37oC for 15' and
: stop with 0.5ul of 0.5M EDTA. Loaded directly on preparative gel and cut
: out the band (band prominent in 0.67u and 0.22u lanes). Best of luck!


: Brett Lindenbach
:     
: Program in Immunology                              
: Washington University - St Louis                  
: brett at borcim.wustl.edu                             




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