Help! Does anyone have a PCR Protocol for BIG Primers (Forward=20mer, Reverse=40mer...Tm is ~83C)

Dandog ddam at
Fri Dec 10 18:51:03 EST 1999

I am performing a PCR derived deletion mutagenesis to develop specific
target and competitor sequence bp fragments. The design is as follows:
[abbrev: Forward Primer = P1, Reverse Primer = P2, Internal Primer = Int,
Deletion Mutagenesis Primer = P3]  My P1 and P2 primers are 20mers and
they produce a product the right length on an agarose gel. My P3 primer
is a 40mer, consisting of P2+Internal primer to delete a 100 bp segment
from the P1 and P2 derived fragment. On other experiments this strategy
has worked using normal PCR protocols with annealing temperatures -2°C
below the Tm. However, I realize the 40mer presents a problem due to it's
large Tm ~83°C and it may not work all the time. Does anyone have a pcr
protocol for large oligo primers? Your help is appreciated!

Danny Martinez, Ph.D.
Connective Tissue Physiology Laboratory
ddam at

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