2 Rounds of PCR
hamel at ccu.umanitoba.ca
Wed Mar 3 16:46:41 EST 1993
In article <9303031439.AA03200 at horvak.hrl.dk> hww at horvak.hrl.dk (Henrik Wengholt) writes:
>Artifact after 2 rounds of PCR
>When the product obtained after one round (30 to 40 cycles) of polymerase
>chain reaction (PCR) is subjected to a second round of PCR with the same
>primers the result is often a smear rather than the expected product. The
>smear as well as nonspecific bands are seen with most primer sets after the
>second round of PCR even without added template (this is probably not due to
>contamination). What is the cause of the phenomenon and how can it be
>P.s. In my case it is not possible to use internal (to the first primer set)
>primers for the second round of PCR.
>Henrik Wengholt email: hww at hrl.dk
>Hagedorn Research Laboratory ph: +45-31-68-08-60
If your PCR product size is less than around 1 kbp, and the reaction
volumes are no more than 100 uL (in 500 uL tubes), then perhaps try shortening
each cycle step to 15 seconds.
1/ 95oC for 15 seconds
2/ 60oC for 15 seconds (substitute temp with Tm of your primers minus 5oC)
3/ 70oC for 15 seconds
Cycle back to step 1 for up to 50 cycles (shorter step times for more cycles).
Why are you subjecting to second round? Have you attempted just performing
one round, comparing for 30, 35, 40, 45, 50, and 55 cycles? Smearing is
quite common if too many cycles are run under "standard" conditions.
Shorter step times and/or less "overload" of primers are but two of
several conditions that can be considered.
You might also consider running between 15 and 25 cycles using about 1/10
amount of primers that you normally use (say about 1- 10 pmoles each primer in
100 uL reaction), then adding more primer (about 100 pmoles) for the
remaining cycles (15- 35 more cycles).
Also, if using Taq pol. then a specificity enhancer may prove useful (such
as from Stratagene). For other thermostable DNA pol. formamide can be
included in the reaction (up to 18% v/v in case of Tth pol.), New England
Biolab's Vent exo- DNA pol. will easilly withstand 10% formamide (18% too
if Mg++ is increased to 4 mM final conc.).
Manitoba Veterinary Virology
email: hamel at ccu.umanitoba.ca
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