Problem converting mutiplex PCR to single reactions

Aug 18, 2017
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#1

Hello everyone! I'm having an odd problem with my PCRs that I desperately hope some of you clever folks can help me figure out. I am looking at a well-known ~600bp fragment of Mitochondrial d-loop region. This fragment amplifies perfectly well when using a Qiagen multiplex mastermix (designed for amplifying more than one fragment). However when I use the Qiagen singlex/singleplex Taq polymerase mastermix(designed for amplifying one locus per reaction), there is absolutely no amplification whatsoever.

I have tried the following variations with the multiplex mastermix(control), singlex mastermix and singlex mastermix with additional MgCl2:

1. A gradient of annealing temperatures with all other steps according to published information for this same fragment

2. Reaction conditions recommended for singlex mastermix

3. Reaction conditions recommended for multiplexing

In the gradient PCR, only the multiplex samples amplified (and did so for all annealing temperatures). In the second PCR, nothing amplified. In the third PCR, only the control(multiplex mastermix) amplified.

I can't figure out why a single locus would amplify only with multiplex mastermix and not with the singlex mastermix. Please help!!!!!!!!!!!!!
 

Ivan

Administrator
Staff member
Nov 16, 2015
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#2

Hi Upasana,

The issue you are having is not unusual. You got a PCR reaction to work using a specific PCR mix and cannot get it to work using a different PCR mix. Since these commercial reagents do not publish the composition of their buffers you cannot really say what is in the first reagent that makes the PCR to work that may be missing in the other reagents that do not work.

My recommendation to you is not to spend too much time troubleshooting this and instead either use the reagent that already works or re-design this assay and see if it works with the other reagents you want to use.

Good luck.
 

Ivan

Administrator
Staff member
Nov 16, 2015
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#4

Yes, you have a problem converting a multiplex PCR to a single PCR. But as I said in my previous post if the multiplex PCR is working why not just run the multiplex PCR? If the single PCR fails then just run the multiplex PCR. Alternatively you may have to re-design the PCR.