Annealing Temp=Extension Temp - (Oct/13/2006 )

Hi! I'm just wondering what happens if you annealing Temp is equal to your extension Temp?

I assume that the annealing of the primers occurs at the same time that the extension, some strands will be annealed and immediately extended. If I desgin a primers with such a high annealing Temp (let's say 68 -many polymerase starts to extend at this Temp or 72) what problems I could face?

Any opinion would be helpful.Thanks.

-gsamsa-

QUOTE (gsamsa @ Oct 13 2006, 12:09 PM)

This is commonly known as "two-step" PCR, where you use cycles of just 2 temperatures like (94°C 30'', 72°C 1'30'' ). Works usually quite well.

Mike

-jadefalcon-

QUOTE (jadefalcon @ Oct 13 2006, 05:42 AM)

This is commonly known as "two-step" PCR, where you use cycles of just 2 temperatures like (94°C 30'', 72°C 1'30'' ). Works usually quite well.

Mike

sometimes we need to do a 2 step PCR and they work perfectly fine. even for sequencing shRNA, we were advised to do a 2 step PCR.

-scolix-

it is ideal condition of PCR

-rye-

Thanks a lot for all replies.

I will keep them in mind.

:-)

-gsamsa-