Problem Converting Multiplex PCR to Singlex PCR

Go to the profile of mealyworm
Aug 18, 2017

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.


Go to the profile of mdfenko
mdfenko 7 months ago

did a control reaction amplify with the singlex mastermix?

maybe a cofactor found in the multiplex mix is missing or reduced or increased in the singlex mix?

Go to the profile of OldCloner
OldCloner 7 months ago

I'd also suspect the singlex kit might have gone bad. Use a kit control as suggested by mdfenko, or try a different supplier's kit, maybe with a proofreading polymerase. Could your template have a contaminant that is neutralized by the special additives in the multiplex kit? Try diluting it/adding less. Is there an alternate target you could amplify from the same template, to see if that works?

Good luck.