I will start using SuperScript™ III to make RT reactions. I have some doubts about the ideal temperature in RT reaction and about the amount of enzyme per reaction. In the protocol, they suggest 50 C and 1 microL of enzyme (but I would like to use less quantity because it is very expensive). Any suggestion about the initial amount of total RNA per RT reaction?
I use SuperScript III for my RT reactions pretty much every day for the past two years with success. Conditions of the RT rxn depend on the quality of your RNA as well as type of your RNA (how structured it is). My typical experiment is set up as follows: first I set up annealing reactions where I add: 5uM of RT primer with ~5 pmol of RNA of interest in total of 12 ul of reaction.
I was successful with as little as 0.25 pmol of my RNA and I was still able to obtain good yield of cDNA products.
I put that initial annealing reaction in PCR block for the following conditions: 85 C for 1 min, 60 C for 5 min, 35 C for 5 min and finally 50C for 50min.
During that time I prepare my RT mix:
5x RT buffer, 9.14ul,
100mM DTT 2.28 ul,
10mM dNTPs 2.28 ul,
SSIII 1ul
1.3 H2O
(total volume 16ul)
I put that mix in heating block for 3min at 37C before I add it to my annealing rxn (which as you remember I set up at the beginning).
When my annealing rxn is at final seconds of the step 35C I add the warmed up RT mix (8ul of RT mix to 12 ul of annealing rxn.
Finally after the rxn is over, I hydrolyze my RNA by adding 1 ul 4M NaOh and incubation for 5min at 95 C. After that I put my rxn on ice for 5 min and neutralize by adding 2ul of 2N HCl. Then, I precipitate the rxn with EtOH and NaoAC, centrifuge and analyze.
Good luck!
Joanna,
I was checking the Script III protocol and I have found some differences with your protocol:
1. The annealing reaction is just 60 5 min and after this 1 min on ice.
2. DNTPs are included in the annealing mix
3. RT mix is not heat
Why did you introduce these changes?
Thanks a lot
I use this protocol for SHAPE experiments, which require very high-quality cDNA products that are subsequently separated during capillary electrophoresis. I found that the following conditions result in the best yield and quality cDNA products. I don't think adding dNTPs to annealing or to RT mix would make a big difference, and my RT mix is not really heated, it is barely incubated in 37 C for 3 min. Try my conditions and compare them with the ones suggested by company. Like I've mentioned results strongly depends on the quality and kind of RNA you're studying. Sometimes I have to adjust the conditions if I am not getting expected results. It all depends...
- Badges
- Science topic