Please contact Center for Plant Genomics (CPG) facility manager Hailing Jin (hljin@iastate.edu) regarding questions or corrections.
REVERSE TRANSCRIPTION
1. Add 1 ng in vitro transcribed RNA (human gene H2, GeneBank Accession #: AA418251) to the RNA sample (500 ng – 1000 ng)
2. Add DEPC H2O to the RNA sample to 29.5 μl
3. Add 0.5 μl 1μg/ul random hexamer and 1 μg/ul poly dT respectively
4. Incubate the mixture at 65°C for 10 min and then put on ice immediately for 5 min. Let it stand at room temperature for 10 min.
5. Add 18.5 μl pre-mixture, which contains 2.5 μl 10mM dNTP mix, 10 μl 5 x 1st strand buffer, 5μl 0.1M DTT and 1 μl RNase OUT.
6. Mix and spin, stand at room temperature for 2 min.
7. Add 1 μl Superscript II RT and mix gently.
8. Spin down and let it stand at room temperature for 10 min.
9. Incubate at 42°C for 50 min, followed by heat inactivating at 70°C for 15 min.
10. Add 1 μl RNase H, mix gently and spin down.
11. Block at 37°C for 30 min.
Take partial of the reaction products and dilute 100 times, for example, take 5ul and dilute to 500ul, to use as a template for the next steps. Store the rest 1st stand cDNA at -20°C.
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