Can we use regular PCR master mix for qPCR assay? Or we should buy qPCR master mix. I am going to use taqman probe for my qPCR assay.
Taqman is a really sensible assay, and the enzyme, MgCl, and other stuffs in proper master mix is optimized for it. Probably to use a regular one, do will spend lots of time (and money) optimizing it.
You can buy a Taqman mix from other company that is cheaper and compatible with your equipment. Keep in mind, that if you will use Taqman probes will you need taqman master mix. If you used a SyBr green mix (useally is cheaper) and taqman probes, you will have two different fluorescent signals and an inadequate result.
Hi..
You cant use regular PCR master mix..
Basically, for detection of qPCR product two common methods are being used
1. Non-specific fluorescent dyes that intercalate with any double-stranded DNA,
2. sequence-specific DNA probes consisting of oligo nucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe
the master mix should contain either of this two, which regular master mix doesn't have.
Sushil I am not clear with your answer.... I will add my probe and primer along with my template at the end only..my master mix wont be having these things...and as i know Master mix contains dNTPs, MgCl2, polymerase, and buffer...so difference is there in regular master mix and qPCR master mix???
I am using Taqman assay ( NOT SYBER GREEN) and my Taqman has one FAM (fluorescent dye) and NFQ (quencher) .... which will give me detection and quantification of specific amplification... so is that necessary to use Taqman master mix for my assay..... what is problem with regular PCR master mix????
Companies don't do nearly as much "optimization" as you think. Just do it, see what happens. You can always go to the ridiculously priced, so-called "optimized" stuff when you know that you have a problem.
I dont think you can.
This master mix is designed especially to optimize the best condition of the primers.
For example, fast master mix is designed especially for fast reaction QPCR
I don´t see why not. The problem will be the optimization, but once it's optimized, it should work well.
If you have a PCR that is working fine with your primers (means a clean band on gel), then probably you can just add the probe to it. Verify the reaction again on gel to ensure that there is no degradation of performance. If you see additional bands or smear, it would need optimization for run conditions.
Before that, check the primers and probe for any potential dimers, hairpins etc using a software (eg: IDT oligo analyzer)
Reviving an old question here:
So did you try it with a normal PCR mastermix?
I'm waiting for my qPCR mastermix to arrive and is wondering if I should just try the PCR mastermix during the wait.
Like Phuvadol i'm also curious if you had succes. I will try with my normal PCR mastermix+primers+templates+tagman-probe, because why not try :) If it doesn't work I just order a prober tagman qPCR mastermix...
If you use a common MasterMix or Taq buffer the amplification will work just as fine as with qPCR MasterMix or qPCR Taq buffer, so if you do agarose gel electrophoresis after the PCR there will be no difference between them.
But there is a difference in the accuracy of the fluorescence lecture, i don't know why but the lecture in the common MasterMix has more noise and background signal, and also, the lectures doesn't seem as good, meaning there won't be a clear sigmod, as with a qPCR buffer. Of course, this could change depending on the buffer you are using. I did it with Standard Taq Buffer (10x), New England Biolabs.
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