Afl II ligations... any suggestions?

Name fred at here.there.com
Tue Mar 3 19:48:04 EST 1998


Dear netters-

I've repeatedly tried to do some cloning using an AflII site...and I've
tried a couple of obvious tricks to get any useful efficiency, but to no
avail.  Specifically, I have a 650 bp. EcoRI/Afl II fragment I'm trying to
get into the same sites of pSL1180  (superlinker from pharmacia).

Production of the fragments goes well, and I even have tried CIP-ing the
vector (just in case...)...I've tried ligation +/- PEG, high and low
amounts of DNA, at 12 degrees C and room temp., and high and low ligase
(400 and 2000 NEB units).  

And all I get are (very few) weirdo clones; that is, clones i can't
explain from fer instance, insertion of a dimer of my insert.  (and these
represent 3x more colonies than without insert!)

Any ideas?


Thanks in advance!!

Micky Krol

Inst. for Molec. Virology

UW-Madison

em-ay-kay-are-oh-ell at students.wisc.edu

(translate the first part into letters to use this address)



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