hamel at ccu.umanitoba.ca
Tue Apr 20 10:31:35 EST 1993
Have you tried diluting the first PCRs before adding them together? (say 1
in 10,000 dil'n), also what about the cycling step TIMES? Perhaps too
short for the elongation step? (say 95oC for 5 min soak followed by say
20, 30, 40 cycles of 95oC/15 sec, annealing temp/15 sec, 70oC/15 sec with
increase after each cycle so that final 70oC step is for, say 2 min, then
finish off with final soak at 70oC for say 5- 10 min)....
In article <1993Apr20.094111.1 at wehi.edu.au> gough_k at wehi.edu.au writes:
>I am trying to splice PCR fragments together using the PCR.
>I have two 1kbp fragments and want to join them togther by PCR.
>The overlap at the ends is between 18 and 30 bp but I am getting
>variable results. Sometimes successful other times not so.
>I have tried various temperatures, enzymes, DNA concentration
>but with little help.
>SOOOOOO HELP. Does anyone have any success?
>Keith Gough, Melbourne, Australia.
>Gough_K at WEHI.EDU.AU
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