Multiplex PCR - tips and hints for optimization (Nov/02/2005 )

Hi all!
I need help. I need to multiplex my CMT STR's test, but I can't amplify some of the fragments. I've read lots of things that I could change but I don't have the time to experiment it all.Could you pleeeeeeaaaaase give me some tips about multiplexing? What is fundamental that I can not miss?

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Well maybe first you gave us more hints on your abbrevaitions and we could help more

Anyway from my experience here are some important factors

1/ Your primers have to be used at the same temperature so you should optimize your Tm to do so

2/ Better select the size of the expected product not too close in size (hard to differentiate) but not too different in size 1.5 Kb takes more extension time (1.5mn) to generate than a 150bp fragment

Pesji

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You should optimize your primer concentrations also for dimers and so they don't outcompete each other

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Hi IMi,

I've been working with multiplex for a year or so and I think the best way is to stablish some standars before starting:

- For me, the mooooost important thing was primer design (not many primer dimers among all the markers and little selfpriming). GeneWalker is a good program in the internet to test them and I designed all with primer3.

- I fixed some conditions in my PCR before I started: MgCl2 concentration (2 mM), primer volume (1 microl from 5 micromolar for each PCR reaction) and all the rest, leaving annealing temperature for the thermocycler.

- I used two samples only (already typed) one was K562, a good standard, and one sample extracted by the method I used for DNA purification (phenol-chloroform for me).

I only changed annealing temperature first and primer concentration. It worked for me and now it's my labs routine!!

Hope some of this helps.

Esther

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To my experience, optimising multiplex PCR takes time! I agree with the previous post that the most important is the primer design. Put much time and effort there!

What you can do if you don't want to do so much experiments is to buy a multiplex optimisation kit. For instance Qiagene has one. In our lab it has been used by a master student, with some promising results.

Good luck!

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