Consistent subcloning from agarose (finally !)

the End jgraham at bronze.ucs.indiana.edu
Mon Apr 26 09:44:28 EST 1993


Paul,

Thanks for reposting some critical points from my post. I suggest that
interested readers grab the entire post from the gopher server at Indiana
"IUBIO".

Experimental data on the inactivation of vectors by exposure to UV
transillumination can be found in P. Hartman. 1991. Biotechniques 11 (6)p.746.
Hartman demonstrates a thousand-fold reduction in trasnformability of plasmids
exposed to 302 nm light for 1 minute. Smaller vectors survived longer,
and some protection was afforded by a "UV-transparent tray" (50%), while
only 360 nm peak bulbs were successful in maintaining plasmid integrity.

I have also had intermittent success with glassmilk preparation of DNA
fragments. I did not however find it to be a reliable procedure.

As with almost all of these techniques, it is the efficiency of the procedure
that is important is giving them wide-scale applicability. While certain
practices may give acceptable results with some combination of vector and 
insert, they may not be sufficient for other more demanding situations.
If procedures can be streamlined without unacceptable reduction in efficiency,
then they may be more suited to an individual application. In the case
of subcloning from low-melting agarose, it is difficult to predict the
difficulty in obtaining a specific construct, and even a 50% reduction
in efficiency would often drop below the useful threshold in my hands.

Good luck,

Jim
J. Graham
Biology and Chemistry Departments
Indiana University -Bloomington 



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