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Odd issue converting mutiplex PCR to single reactions

PCR multiplex PCR Multiplexing PCR methods PCR conversion

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#1 MealyWorm

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Posted 18 August 2017 - 07:54 AM

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!!!!!!!!!!!!!



#2 bob1

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Posted 18 August 2017 - 12:48 PM

The difference will be in the components of the reaction mixes - check the product information for buffer components and see what is different between the two. Also check the amount of DNA in the reaction - multiplex reactions typically require more DNA than single reactions which can sometimes inhibit single reactions.



#3 MealyWorm

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Posted 19 August 2017 - 01:07 AM

Thank you bob1! The only difference I could make out was that the mulitplex mastermix had double the amount of MgCl2, ad thus added more MgCl2 to the singlex mastermix in the trials. I will try the PCRs again with a reduced amount of DNA.

 

The difference will be in the components of the reaction mixes - check the product information for buffer components and see what is different between the two. Also check the amount of DNA in the reaction - multiplex reactions typically require more DNA than single reactions which can sometimes inhibit single reactions.



#4 mdfenko

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Posted 21 August 2017 - 04:24 AM

also check if they use the same polymerase


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