Methods Digest, Vol 24, Issue 9

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Tue May 15 07:44:20 EST 2007


> To: Plasmid construction


I think problem is not wrt compatibility of ends. In any case if the insert
is good and intact it must ligate. The problem is with the tiny amount of
insert DNa what you are getting after cleaning from the gel. Even the tiny
amount of DNA should be enough for successful ligation. The problem seems to
be with self ligation of the vector ends generated by BamH1/ BglII. One
solution is treating cut vector with CIAP to prevent self ligation. If this
is not working, surely ends of vector and/or insert are being chopped off by
some exonuclease contamination in the say elution buffer or TE Buffer. I
have been successful in getting enough DNA purified from a single band for
subsequent ligations and have also failures and the culprit has alwys been
purity and storage conditions and age of TE and water. Overexposure of
agarose gel while checking the DNa or cutting the DNA bands also results in
such chopping of end nucleotides.
Try cutting higher amount od DNAs, run immediately, cut desired bands as
fast as possible using low intensity of UV, purify DNA without giving any
break and proceed with ligation. treat vector and insert similarly. use
freshly prepared TE and water that has been recently sterilised.You can also
autoclave small volume of elution buffer/ 1/10TE for the purpose. using
higher ratio of insert/ plasmid DNA that have been dissolved in normal TE
also results in low ligation efficiency due to interference by EDTA (use
10mM tris instead.
All the best
Dr V K Gupta
Insect Molecular Biology Lab
PAU, Ludhiana


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