AmpliTaq Gold instead of TaqMan Universal PCR Mix in Real-time? - (Aug/27/2005 )
Hi every one..
I am new to the real time PCR. I want to do relative gene expression using the TaqMan Gene Expression assay reagent from Applied Biosystems in which they supply the primers and probe mix.
Applied Biosystems says that we have to use their primers-probe mix along with their TaqMan Universal PCR mix that contains the AmpliTaq gold enzyme along with MgCl2 and other PCR buffer components that they don't disclose. This mix is too expensive.
My question is, can we simply use the AmpliTaq Gold polymerase with buffer II and to optimize the MgCl2 concentration ourselves or not?
If some one has any experience with it please I really need help in this. I need to finish my work in this that is part of my PhD project. I don't have much money to waste neither time to order things and they don't work.
Waiting for replies.......
The nature of the Taqman assays requires the Taq polymerase you are using to have 5' exonuclease activity to digest the probe so that the fluorphore and quencher are separated and thus you get fluorescence. Though I have not tried using another Taq, I can't see it being a problem to use a home made master mix with a Taq with 5' exo activity in a TaqMan assay. You may need to optimise a bit, to get the assay working properly.
It's a juggle between saving some money (in this case, lots of money) and getting the results in good time. TaqMan reagents are expensive but are guaranteed to work.
Food for thought.
It should work with AmpliTaq without problems. But if you will not add ROX to the mastermix, you should be very careful with pipeting and bubbles.
sybr green is way cheaper and works well, you just have to engage your brain when designing your primers
definitely don't forget the ROX, I don't care how well you pipette there are always very tiny discrepancies and these will be vastly magnified in qPCR