PCR Supermix vs Roche - (Jul/27/2005 )
Just have a question about PCR
I used to use PCR supermix from invitrogen, but recently my friend had told me that Roche PCR system works better, so I used it.
Well, and I actually run agarose gel, with two samples, (one amplified by supermix, and other was amplified with Roche kit)(of course, same DNA sample)
And result was quite different between these two.
Could someone explain how the result can be so different?
Am I doing something wrong?
I don't think you are doing anything wrong. There are a number of reasons for different Taq/mastermixes to work differently. I am not familiar with either of the systems u are using, so I cannot speak in specifics, but some differences that could affect your result are:
The taq enzyme, its fidelity and rate of processing. Even the exact same taq can work better or worse in different preparations of the same enzyme.
Magnesium concentrations may differ
other additives in one reaction not in other.
This is one reason that internal controls are so important. The above should affect amplification of your gene of interest as well as the internal control the same. I say use the one that gave you the best amplification(clean, most amount of product)
There indeed is a differnece between different enzymes/buffers. Magnesium concentration is probably one of the most important factors to consider. Different enzymes have different optimal magnesium concentrations, and this will affect annealing of your primers. So, if you want to really compare 2 enzymes, you have lot of work to do, trying different annealing temperatures/different Mg concentrations (most enzymes come with tubes of MgCl2 or MgSO4 so you can adjust your concentration).
I myself once checked an enzyme that uses MgSO4 for how it works with MgCL2 and found a huge difference in yield.
You can't really predict too much when trying new enzymes, trial and error is the way to go.
I have found that the most important issues (primer design, annealing temperature) are hard enough to optimize. PCR reactions have dozens of variables. Especially in the beginning, I would highly recommend that you choose one reputable manufacturer's enzyme, and spend your time optimizing other parts of your reaction. If you bounce around with different enzymes, different buffers, different additives, and different voodoo dances, you'll never figure out what is really going on, and why it works today and not yesterday. There is time later to expand your choices, when there is good reason (long templates, high GC, A tailing, strand displacement, very high fidelity).