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Thermal Cycler for Multiplex PCR

thermal cycler multiplex pcr

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#1 burakkc

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Posted 04 March 2016 - 04:05 AM

Hello,

I have a quick question. Do I need a real-time cycler for multiplex pcr analysis? Can I just simply mix the primers and run them on a conventional cycler? PCR products will be analyized using ABI 310 squencer.

 

Thanks,


Edited by burakkc, 04 March 2016 - 04:06 AM.


#2 phage434

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Posted 04 March 2016 - 06:00 AM

In principle this should work fine. In practice, multiplex PCR is often quite problematic, since you need to optimize two different PCR reactions at the same time. If you are willing to devote some time to optimization, then this is a worthwhile effort. You sequencing, of course, would need to be with each product independently.



#3 burakkc

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Posted 04 March 2016 - 06:04 AM

I have access to a real-time cycler that is in another lab in our university, but I can't consantly use it. Maybe I can use it a couple of times to optimize pcr and than run the rest on our cycler? Would that work?



#4 Trof

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Posted 06 March 2016 - 06:17 AM

You obviously need to do only classic PCR, and the product will be separated and assessed by the fragmentation analysis. That means primers are tagged with fluorphore, right?

Just how many-plex are we talking here about?

 

And for your question, no, you don't need a real-time PCR for that. If you have all primers tagged with FAM (which is likely), you would be able to see it on real-time cycler,but there is no point in that, if all are tagged by same fluorophore, there is no way to tell them apart on real-time machine.

 

For use on the ABI analyser, you need to optimize the dilution of well-runing PCR to fit into fluorescence levels of the analyser.

 

So, If you are new to this assay, I would first test each primer (if possible) alone, to get a good product (so you see they work fine), then optimize multiplex (this can be tricky, but you can google what you can do, mainly by tweaking reaction conditions and the PCR program), if it looks good, put several dilutions on ABI analyzer and see, where do you hit the right fluorescence level. Usualy PCR product is used pretty diluted for fragmentation analysis.


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