Primer design and need help - (Feb/12/2009 )
zack on Feb 12 2009, 09:45 AM said:
who about denaturation 94- 1 min... should i change it to 30 secs too
yes. Reduce that too. 30sec would be fine. But i think you can go down to 20sec.
I would also reduce the number of cycles to 25. Taq isn't a proof reading polymerase and makes a fair number of mistakes. It isn't something you usually want to use in a cloning. Furthermore, the PCR reaction does peak.
Also at around 30+ cycles, the amount of PCR product produce levels offs.. due to inhibition from pyrophosphate.
It is more efficient (time) and safer (fewer mistakes by the polymerase) to run two tubes of the PCR reaction than try to push a single tube to high yields to obtain enough product.
-perneseblue-
perneseblue on Feb 12 2009, 09:53 AM said:
zack on Feb 12 2009, 09:45 AM said:
who about denaturation 94- 1 min... should i change it to 30 secs too
yes. Reduce that too. 30sec would be fine. But i think you can go down to 20sec.
I would also reduce the number of cycles to 25. Taq isn't a proof reading polymerase and makes a fair number of mistakes. It isn't something you usually want to use in a cloning. Furthermore, the PCR reaction does peak.
Also at around 30+ cycles, the amount of PCR product produce levels offs.. due to inhibition from pyrophosphate.
It is more efficient (time) and safer (fewer mistakes by the polymerase) to run two tubes of the PCR reaction than try to push a single tube to high yields to obtain enough product.
okey.. thank you ...i will follow ur advice .. then i will give the result soon as possible
-zack-