Ligation Problems

Eric C. Anderson anderson at pharmdec.wustl.edu
Thu May 25 10:21:40 EST 1995


In article <3pu1a0$5cg at quandong.itd.adelaide.edu.au>, Sam Hodge
<samiam at smug.student.adelaide.edu.au> wrote:

> Dear Reader,
>                    Our lab has been having difficuties ligating PCR products 
> into vectors. The vector has been fully digested and then gel purified 
> using a glass bead kit. The PCR product has been cut ( or we think it 
> has) with two different enzymes. It is difficult to know if the PCR 
> product has been fully digested as only a few nucleotides have been 
> removed from the PCR product. 

the biggest question is whether you've actually cut it or not.  what
enzymes are you using to cut with?  some enzymes cut very well at the ends
of DNA and others don't cut at all, e.g. when an EcoRI site is within 8
bases of the end, it will cut with greater than 90% efficiency in a 2h
digest.  when HindIII is within 10 bases of the end of a sequence however,
it won't even cut in a 20h digestion.  for more info on which enzymes cut
close to the ends of sequences, check out the New England Biolabs Catalog,
pp.208-209 (for the 1995 catalog anyway).

good luck,

eric

-- 
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson                                 anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)362-7058 (FAX)
660 s. euclid box 8103                           
st. louis, mo 63110

for a comprehensive list of bike related web pages... http://pharmdec.wustl.edu/~anderson/anderson.html



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