Hello,
I am working on developing a real time RT PCR based kit. For that, I need to prepare 2x one step RT PCR mastermix. I am using PCR Reagents and Verso cDNA synthesis kit from thermo scientific. I have used all the PCR reagents (Including 10x buffer, 25mM MgCl2, 10mMdNTP mixture, taq DNA polymerase (5U/ul) and nuclease free water) and only the reverse transcriptase enzyme mix and RT enhancer from the verso cDNA synthesis kit. For a 100ul reaction the composition used was -
10x buffer= 20ul (2x)
25mM MgCl2= 3ul
10mM dNTP= 2ul
Taq polymerase= 1ul
Reverse transcriptase= 5ul
RT enhancer= 5ul
Water= 64ul
No amplification curve was seen when the real-time PCR was run. Now i am confused about if i need to add cDNA synthesis buffer in the mixture or any other component from the cDNA synthesis kit? and which PCR protocol i need to follow (the one for taq polymerase and another for reverse transcriptase enzyme)
As far as I can see there is no intercalating dye such as SYBR Green present in the mix (if it is not included in the 10x buffer). In this case there can be no generation of a signal (emitted green light) even if the reverse transcription and subsequent PCR are successfull.
You are using the incorrect amounts of the reagents.
For example, in 100 microliters of total volume, you need to use 10 microliters of buffer.
And unless your buffer includes a fluorescent dye, there is no way for the qPCR machine to detect any product.
Just buy a one-step kit and/or find a protocol that you know will work - do not design your own.
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