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....
Sounds like it should work, as long as the polyermase is the same PlatinumTaq and not a specially formulated one to work alongside the RT.
Unless you're super lucky and hit on the optimum conditions/spend ages optimising you might not get as good yields (complete lack of chloride hints that the enzymes might be fussy), but given how expensive these kits are just for spare buffer I'd say it's definitely worth a shot!
Best of luck!
go ahead john infact, thermo is very very and only business oriented, its surprise they do same on the other side of globe too. There is no harm in doing a reaction with your home brewed buffer, since your buffer can be also diluted to achieve optimicity it can be very well be tried
Well the 1-step RT-PCR kit suggests that if you want to do no-RT controls (which they don't give you enough buffers for) you should substitute the kit's enzyme mix for separately-purchased PlatinumTaq polymerase. So it would be a bit cheeky if they weren't the same enzyme. And I have actually had DNA contaminations show up in these no-RT controls in the past with the separately-purchased polymerase used with the kit bufer, so clearly the separately-purchased polymerase is compatible with the RT-PCR kit buffer.
I feel like you're totally correct Frank. On a totally unrelated note, I'm not sure whether Thermo can take any action against someone publicising their "secret" buffer recipes overtly online. In the totally hypothetical situation that one might do such a thing.
Hi Laura,
I've not tried making this buffer, but I know someone who has & it seemed to work perfectly well.
Just to clarify for everyone, yes I did try out that buffer composition, and yes it works as replacement 2X buffer for the SuperScript III one-step RT-PCR kit.
Also, I'm clearly not the only person to have guessed the buffer composition (or at least close enough) since Thermo now include more of the buffer in the kit.
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