4 replies to this topic

[icarus.rachel]

Hi all,

It is about running the multiplex PCR. When I did the PCR separately, the band can be run individually. However, when I mix two primers set together, and half the total vol.. I cannot see any bands in the gel. Could you please offer any suggestions so that to improve? Thank you.

[Adrian K]

icarus.rachel, on Dec 1 2009, 03:55 PM, said:

Hi all,

It is about running the multiplex PCR. When I did the PCR separately, the band can be run individually. However, when I mix two primers set together, and half the total vol.. I cannot see any bands in the gel. Could you please offer any suggestions so that to improve? Thank you.

Did your primers self-binding together? (Primer Dimer)
Tell us more of your protocol so that we can help you more...

[Maddie]

Tell us also what the annealing temperature was for each primer and the one you used with your duplex.

[icarus.rachel]

adrian kohsf, on Dec 1 2009, 04:16 PM, said:

icarus.rachel, on Dec 1 2009, 03:55 PM, said:

Hi all,

It is about running the multiplex PCR. When I did the PCR separately, the band can be run individually. However, when I mix two primers set together, and half the total vol.. I cannot see any bands in the gel. Could you please offer any suggestions so that to improve? Thank you.

Did your primers self-binding together? (Primer Dimer)
Tell us more of your protocol so that we can help you more...

I got the primers, 402bp (Tm: 66-77C), 308bp (Tm: 76C) & 245bp (Tm: 77C). with annealing 58C for 30s (according to protocol).
If I decrease the annealing temp, how low should i adjust?
if i decrease PCR primer & increase the template? but they work when do it in separate.
what about increase MgCl?

Thank you very much in advance.

Edited by icarus.rachel, 01 December 2009 - 07:58 PM.

[Adrian K]

Dear icarus.rachel,

From what you mention, I suggest you don't cut down the concentration for each primers, add DMSO and do a gradient PCR. Make sure your total primers concentration is not more than 2mM.