I do a lot of RT-PCR and always include a no-RT control (without reverse transcriptase) for each of my samples, so that I can rule out DNA contamination in my RNA samples. Unfortunately, the SuperScript III 1-step RT-PCR kit (with enzyme mix of SuperScript III reverse transcriptase and Platinum taq polymerase) does not come with enough buffer to allow for this, which is a shame as this kit is very expensive. I contacted Thermo and they told me that they cannot sell me spare tubes of buffer and also cannot give me the composition of their secret optimised reaction buffer. This is a shame, though I totally understand why they would want to maximise their profit margins by pretending that their kits contain enough buffer for 50 reactions when in reality, they don't.

This led me to wonder if it isn't possible to guess the buffer composition and try to make it myself. The one hint that is given on the product data sheet is that the 2X reaction buffer contains 0.4 mM of each dNTP and 2.4 mM MgSO4. I also know from the information given with the Platinum taq polymerase what the composition of that reaction buffer is. I therefore wonder if one couldn't make the 2X reaction buffer for the kit by simply making a buffer containing 120 mM Tris-SO4 (pH 8.9); 36 mM (NH4)2SO4; 2.4 mM MgSO4; and 0.4 mM of each dNTP (clearly the platinum taq polymerase is very sensitive to chloride ions and the SuperScript III reverse transcriptase requires magnesium ions). I just have this very strong feeling that if one were to make that buffer, it would work very well indeed with the SuperScript III one-step RT-PCR kit as a replacement for the 2X reaction buffer, and could be very useful for making sure that the one gets the most out of this very expensive kit. I'm not sure how Thermo would feel if someone were to try that and publicise it on the internet, but somehow I feel like that recipe would indeed work very well....