long pcrs

brett at BORCIM.WUSTL.EDU brett at BORCIM.WUSTL.EDU
Thu Apr 7 09:43:42 EST 1994


hello, someone recently raised the question of whether long pcrs ala klentaq
are actually amplifying or not, because wayne barnes starts with 10 ng template
which is much more than the 1 ng one normally uses with taq. i forgot who
wrote this and the mail file has already been deleted, but may i try to
address this? if one started with 1 ng of a 2kb fragment, you would have
approxiamtely 0.75 fmol DNA. 10 ng of 35kb is roughly 0.43 fmol of DNA. Thus,
you are starting with less DNA, on a molar basis. However, most templates are
not fragments whose whole length is amplified. ie, one might use 1ng of a 10kb
plasmid as template for std PCR, so interpret this as you wish. I have used
wayne's enzyme with 1 ng plasmid DNA for doing amplifications of around 4kb
with excellent results, but am not in the business of amplifying whole phage
genomes. brett at borcim.wustl.edu



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