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Multiplex PCR troubleshooting - (May/13/2005 )

Hey,
G'Day
I'm working on Multiplex PCR using 6 different primers with 6 serotypes of the bug. But doesn't seem to be working sad.gif . Have followed the protocol from the literature but hasn't been helpful blink.gif pls....

-caala01-

Are the primer sets working in singleplex?

-spotty909-

Multiplex PCR can be tricky!
I've found that the annealing temperature can be crucial! I've tried a multiplex PCR where you'd only get the three bonds on the temperature 56.3 C... A decimal above this temperature, only two where visible.

It's also a good idea as already mentioned to check the primers, but also the polymerase.

A very good troubleshooting guide can be found at http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html

-k_josefin-

QUOTE (spotty909 @ May 13 2005, 03:19 AM)
Are the primer sets working in singleplex?

Thnkx for the reply,
yeap it's working on my singleplex (primer mix and using 1 DNA template). Is there any more protocol for multiplex,
thanks heaps rolleyes.gif

-caala01-

QUOTE (k_josefin @ May 13 2005, 03:45 AM)
Multiplex PCR can be tricky!
I've found that the annealing temperature can be crucial! I've tried a multiplex PCR where you'd only get the three bonds on the temperature 56.3 C...  A decimal above this temperature, only two where visible.

It's also a good idea as already mentioned to check the primers, but also the polymerase.

A very good troubleshooting guide can be found at http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html



Hi Josefin,
I had a look through the Multiplex Trouble site and changed few parameters, but still my Multiplex PCR doesn't seem to be working. Any suggestions is appreciated,
thanks

-caala01-

1. Have you check the location of those primers you use in the genome? The annealing places of the primers are critical to having and making good product
2. depends on how long those amplicons of each are, too long amplicon may cause the multiplexPCR haust,since it has to synthese more targets in the same time.
3. the strategy for optimalize multiplexPCR is begin with two primerpairs,of which both have a colse Tm, optimalize the Tm of the mPCR. You will be happy with this starting stap.
4. I prefer to limit to a TriplexPCR instead of six in one reaction. Since the optimalization of hexaplixPCR seems to me impossible. wink.gif (Take it easy guy, you don't have such hurry like that, just enjoy the labworks cool.gif )

Succes!

-Ribosoul-

QUOTE (Ribosoul @ May 27 2005, 02:09 PM)
1. Have you check the location of those primers you use in the genome? The annealing places of the primers are critical to having and making good product
2. depends on how long those amplicons of each are, too long amplicon may cause the multiplexPCR haust,since it has to synthese more targets in the same time.
3. the strategy for optimalize multiplexPCR is begin with two primerpairs,of which both have a colse Tm, optimalize the Tm of the mPCR. You will be happy with this starting stap.
4. I prefer to limit to a TriplexPCR instead of six in one reaction. Since the optimalization of hexaplixPCR seems to me impossible. wink.gif (Take it easy guy, you don't have such hurry like that, just enjoy the labworks  cool.gif  )

Succes!



G'day,
Thanks for the info. Yeh i've been trying triplex Multiplex where i've mixed 3 primers set and 3 template set in each reaction. In one reaction the longer product. seems not showing up and in another reaction the lower base pair product seems not working!! I've tried changing the conditions and concentrations but still no success!!
Any suggestions from ure experience,
many thanks

-caala01-