CDNA synthesis

From Genome@Yale

Superscript III Information

Protocol for Superscript II

1. Precipitate 20-50ug RNA.
2. Add 3M NaOAc (add 1/40 volume if the RNA is in 75% EtOH).
3. Freeze at -80°C. Centrifuge 15 minutes @ maximum speed.
4. Wash with 500uL 75% EtOH. Centrifuge 5 minutes @ maximum speed.
5. Resuspend in 20uL dH2O
6. 2uL anchored oligo dT + 2uL 10mM dNTP
7. 8uL 5X first-strand buffer + 4uL 0.1M DTT + 1uL RNasin (for 10 reactions: 80uL 5X, 40uL DTT, 10uL RNasin)
8. 65°C for 5 minutes
9. 2uL Superscript RT
10. 1 hour at 42°C
11. 15 minutes at 72°C
12. 1uL RNase A, 1 hour at 37°C
13. Qiaquick PCR purification.
14. OD and normalized [cDNA] to 10 - 50ng/uL.