PCR Subcloning with restriction sites

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Wed Mar 29 04:58:57 EST 2000


In article <38E0C785.1DE75719 at mesastate.edu>, Tony Schountz
<tschount at mesastate.edu> writes
>This is rather interesting. What do you think accounts for the requirements of
>several nucleotides 5' to the restriction site?

Think along the lines of how does an RE locate it's recognition site.
Does it just bind DNA anywhere and then slide/move along until it finds
the site. Which direction does it move 5'-3' or 3'-5'? If one looks at a
xtal structure for say EcoR I complexed with an oligo containing it's
recognition site, how many extra bases outside the RE site are involved?

I should probably know this having worked commercially with RE's for
years, however I kind of worry more about finding, identifying and
purifying them rather than exactly how they work at the molecular level.
Too many other things to know first!

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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