Consistent Subcloning 96 (Re: Freeze & Squeeze)

James Graham graham at biodec.wustl.edu
Sun Jun 16 13:05:03 EST 1996


The Quan and Wilkinson technique mentioned is the key to highly
reliable consistent subcloning when both vector and insert are
so purified. All other methods of purifiying DNA from agarose, while 
perhaps more aesthetically attractive, invariable result in an 
occasional unacceptable degree of sample loss. In the Q&W procedure, 
any two appropriate EthBr stained slices representing vector and insert 
may be merely frozen for 15 minutes and spun. 3 ul of the vector slice 
supernatant may then be ligated to 0, 1, and 4 ul of insert slice 
supernatant in a final volume of 10 ul using a ligase buffer with PEG 
(eg. BRL's).

If one has frozen competent cells tested and stocked (use the Inoue
low temperature grown Ca-Mn-Pipes method in N2) then if you can see
the two bands on a gel, you will obtain your clones, EVERY SINGLE TIME.
I often suggest this regime, and inevitably attentions are caught
by some organic extraction, glass-binding or ion exchange matrix or 
other and progress slows down to a hit or miss speed. The beauty of Q&A 
is there is absolutely nowhere that your sample to go - only one tube is 
used. 

(Dilution prior to freezing is not necessary with low-melting agarose.
Long Wave UV (300+ nm) MUST be used to visualize bands. CAUTION: DNA 
so isolated cannot be precipitated from these supernatants efficiently 
without a butanol extraction and the use of glycogen as co-precipitant.)

> Quan and Wilkinson (1991), Biotechniques, vol. 10, p738.

Jim
J. Graham PhD
Biology Department 
Washington University of St. Louis 



More information about the Methods mailing list