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!

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

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