Alternative to cloning PCR-fragment ...RESULTS
rfwhittier at hotmail.com
Tue Jun 18 22:20:29 EST 2002
Trond Erik Vee Aune wrote:
>The results were as follows: Positive control (1 mikrol LITMUS28
>transformed into my competent DH5alpha) : carpet Negative control (only
>template) : 1 colony Reaction 1 (50 degrees annealing): 10 colonies
>Reaction 2 (55 degrees) : 8 colonies Reaction 3 (60 degrees) : 4 colonies
DH5alpha is recA1 (recombination deficient) host. I realize that
this is standard for transformation of ligated constructs, but
you are carrying out overlap extension PCR which will tend to
produce linear tandem repeats of your target construct.
If your construct does not carry long internal repeats or extensive
homology with the E. coli chromosome, a recA+ strain should work
far better for your purpose, especially since you require efficient
construction of a large library. Recombination can then regenerate
replicative circles out of your linear construct.
If PCR itself isn't working, you'll have to resort to some LA mixture
which combines both an archaeal proofreading (e.g. Pfu) and non-archaeal
(e.g. Taq or Taq derivative) polymerases. These mixtures tend to
be about 3-fold more mutation prone (depending on conditions and
mixture ratios) than proofreading enzymes alone, but if you have
to carry out more than 5 cycles, something is very wrong, and all
the extra cycles will have the same effect of introducing unwanted
Please let this group know what finally works for you.
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