Safety information - Eurogentec
Safety information - Eurogentec
Safety information - Eurogentec
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10<br />
Reverse Transcriptase reaction<br />
5. Thaw the MiRmaid RT primer MasterMIX (red cap) completely and put it on ice.<br />
Vortex briefly and make a short spin by centrifugation.<br />
6. Add 15 μl MiRmaid RT primer MasterMIX to the RNA/DNase treated tube (from step 4).<br />
7. Incubate 5 minutes at 80 °C.<br />
8. Incubate 5 minutes at 60 °C.<br />
9. Incubate 10 minutes at room temperature.<br />
10. Prepare a RT reaction mix by combining the following components, in order, as<br />
detailed below.<br />
COMPONENT VOLUME<br />
5x first strand buffer 9.6 μl<br />
DTT 4.8 μl<br />
dNTP (10 mM) 1.2 μl<br />
RNase free water 5.9 μl<br />
Total mix 21.5 μl<br />
11. Add the RT Reaction mix to RT primer MasterMIX + RNA tube (from step 6).<br />
12. Incubate 2 minutes at 50 °C.<br />
13. Add 1.6 μl of Superscript III.<br />
14. Incubate for 60 minutes at 50 °C.<br />
15. Purify the product with the QIAquick Nucleotide Removal Kit and elute with 40 μl<br />
of EB buffer.<br />
16. Add EB buffer (+/- 285 μl) to the eluate so that the final volume of cDNA is 325 μl.<br />
4. Quantitative qPCR reaction<br />
1. Prepare a qPCR reaction mix by combining the following components in order as<br />
detailed below. It is recommended preparing a volume of reaction mix 10 % to 15 %<br />
greater than required for the total number of reactions (please find an example for<br />
96 reactions also in the table below).<br />
Manual for MiRmaid miRNA precursor qRT-PCR Primer Set I www.eurogentec.com I info@eurogentec.com
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