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multiplex PCR


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4 replies to this topic

#1 icarus.rachel

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Posted 30 November 2009 - 11:55 PM

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. :P

#2 Adrian K

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Posted 01 December 2009 - 12:16 AM

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. :P


Did your primers self-binding together? (Primer Dimer)
Tell us more of your protocol so that we can help you more...
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

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#3 Maddie

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Posted 01 December 2009 - 08:31 AM

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

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Posted 01 December 2009 - 07:54 PM

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. B)


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.


#5 Adrian K

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Posted 02 December 2009 - 06:36 AM

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.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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