4 replies to this topic

[American3pt14]

I was hoping that someone could shed some light on a couple of questions regarding creating PCR overhangs:


1) When calculating what melting temperature to use for the PCR reaction, how should one take into account the overhang (since the first amplification will not use the overhang while all the subsequent amplifications will)?

2) In general, what is the limit of how many base pairs one could create with an overhang and still have a reasonable success rate with PCR reactions (e.g. can a 10 bp overhang work?)

Thanks!
-Albert

[laurequillo]

American3pt14, on 22 September 2010 - 09:20 AM, said:

I was hoping that someone could shed some light on a couple of questions regarding creating PCR overhangs:


1) When calculating what melting temperature to use for the PCR reaction, how should one take into account the overhang (since the first amplification will not use the overhang while all the subsequent amplifications will)?

2) In general, what is the limit of how many base pairs one could create with an overhang and still have a reasonable success rate with PCR reactions (e.g. can a 10 bp overhang work?)

Thanks!
-Albert

Here there are a lot of people that know better than me, but, anyway...
No, for the temperature you just take in account the matching region, and yeah you can add 10 bases (for example a kozak sequence, a restriction site and 3more bases to help cutting)

Edited by laurequillo, 22 September 2010 - 11:46 AM.

[American3pt14]

Thanks-- so just to clarify your 1st point:

Let's say I have a primer xxxxxxxxxxYYYYYYYYYYYYYYY where "x" = overhang and "Y" = matching base pairs.  For the melting temperature then I should only take into account the "Y"'s?  What I don't quite understand is that after the first round of amplification, "x" will have a complement, so the melting temp will increase--so shouldn't that factor into the protocol?

-Albert

[GNANA]

ya u r right, but i think only for the first round of amplification a primer needs more stringent condition, for further rounds it will have enough templates tat can be easily accessed, so u neednt hav to provide more stringency thereafter.

Gnana..

Edited by GNANA, 22 September 2010 - 01:38 PM.

[bob1]

The other posters are correct, you only need to take into account the complementary sequence, ignoring the overhang.  Overhangs can be quite large, I have successfully amplified primers with 40+ bp overhangs while adding tags such as FLAG or HA to sequences.