how can I carry out this PCR profile - (Oct/23/2006 )
Hi everyone, I am new to here and English is a foreign language for me , so any help would be appreciated.
I dont know how to carry out this PCR profiles, espeically the "a further 30cycles"?
"The PCR cycling profile was as follows: 96 °C for 2 min, 10 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. A further 30 cycles were included with the annealing time altered to 40 s and a 5 s extension after each cycle. and then 10 min extension at 72°C."
This is poorly worded, so it is not your fluency in English which is the problem. I interpret this as:
A normal PCR reaction with an initial denaturing of 2 minutes at 96C.
This is followed by 10 cycles consisting of denaturing at 94 C for 30 s, annealing at 55C for 30 s, and extension at 72C for 1 minute.
This is followed by 30 cycles with a 94C denaturing for 30 s, annealing at 55C for 40 s, and extension at 72 C for 1 minute. For each of these cycles, the length of the annealing time is increased by 5 seconds, so the first time it is 40s, the second time, 45 s, the third 50s, etc.
Finally, at the end, there is an extension at 72C for 10 minutes.
Now, that is how I interpret the English, but it makes little sense scientifically. More plausible scientifically, but less plausible from a wording standpoint, would be for the *extension time* to be increased by 5 seconds each cycle. Even that is a little questionable from a scientific standpoint. As I said, this is very poorly worded.
I think phague434 is right. The purpose of the double-cycling (first 10 cycles and then 30 cycles) could be for increase the concentration of the template in the reaction and then amplificate it even more. The other modifications could be for assure the annealing of primers and the extension of the product. The last 10 min of amplification is to amplificate all the PCR-product that could be obteined in that reaction. I think it's very noisy from a technical standpoint: you don't know the annealing time neither extension time. With all that modifications is probably to obtain artifacts from the PCR. Anyway, good luck!
I think you have given me a very clearly explain of this problem.
I can begin my work with no doubt.
Thank u very much.
You're welcome. Regards from Mexico.