Long PCR primers
rafael at howard.genetics.utah.edu
Mon Oct 21 13:42:37 EST 1996
On Mon, 14 Oct 1996, Andrew J. Doherty wrote:
> HI every one
> I've got a PCR primer which is 76 nucleotides long, containing the c-myc
> epitope tag and three overlapping restriction sites but only 12
> nucleotides at the 3' end are complementary to the sequence I'm trying
> to amplify. Is this a feasible primer to use?
Sure it is. I have been using oligos from 40 to 120 bp for PCR for years.
But you will need some tricks, because the efficiency is lower than using
I always gel purify oligos bigger than 30 bp. (check Maniatis).
As you mention, you may start the PCR with 10 cycles at low
annealing temperature; after that, 20 cycles to higher temperature.
Choose a couple of degrees below the melting temperature (calculated with
any method) of the sequence homologous to the template for the first cycles.
Add 5-10 degrees in the following cycles.
Also, you may raise the Mg++ concentration in the PCR buffer. It is so
easy and it gives amazing results. I have found, too, that increasing the
concentration of the big oligos by 5X, the efficiency is also better.
E-mail me for more specific questions (take address from the signature).
Rafael Maldonado | 'No te creas todo
room 6160 Eccles Institute of Human Genetics | lo que leas en
Department of Human Genetics | internet.'
University of Utah |
Salt Lake City, Utah 84112. USA. | "Don't believe
Rafael.Maldonado at genetics.utah.edu | everything you read
Rafael at howard.genetics.utah.edu | in internet."
Tel: 801-581-4429 |
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