help needed with three fragment ligation

Colin Herd c.herd at ion.ucl.ac.uk
Wed May 10 09:53:59 EST 2000


Is there anybody out there who may be able to help me? (don't worry this
is a question about molecular biology, I go to, alt.rec.personalprobs,
for other types of help:-)
    I have a puzzle with regard to a ligation I am trying to perform. I
want to ligate three pieces of DNA together, one of 7.1kb, one of 3.6kb
and one of 1.9kb, to form my complete plasmid for bacterial
transformation.All the fragments use different sticky ends to minimise
problems with the pieces religating in the wrong orientation/order but
it doesn't seem to help. I have done the reaction a number of times
without success.
I would be eternally grateful to anyone out there who has any bright
ideas/tricks of the trade that you have used for ligating multiple
fragments of DNA.
Thanks in advance
Colin






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