length limits of mutagenic primers

Monika Konopka mkonopka at totalise.co.uk
Sun Aug 6 14:15:05 EST 2000

I'm gonig to start using the Quick Change Site Directed Mutagenesis Kit from
Stratagene and my primary concern is the design of mutagenic primers.  Apart
from a few point mutations I'm planing to make some multiple ones. Melting
temperature of my primers with single mismatches is 78 C ONLY WHEN the
primers are AT LEAST 44-46 bp long. For double mutations the primer length
has to be at least 51 bp in order to meet 78 C temperature requirement.
Consequently, the primer length increases with the increase of the mismatch
area and for a 6 aa mismatch (18 bp) the primer has to be as long as 72 bp
to get the melting temperature of only 68 C, which is still 10 C below the
recommended one (78 C). To get the temperature of 79 C my primer should
be... 109 bp long:)..

So, my questions would be as follows: 1. what are the primer length limits
(are primers around 100 bp allowed at all??), 2. what is more crucial for
the mutagenesis efficiency: accurate melting temperature  (78 C) or the
lenght of the primer (not higher than 45 bp) and finally, does the desired
mutation have to be EXACTLY IN THE MIDDLE of the primer sequence or,
possibly, could the difference of 3-8 bp (even up to 40 bp in case of my
multiple mutation) in the length of the two flanking (complementary)
sequences be allowed.
I should be grateful for some advice.
Monika Konopka

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