cDNA synthesis using SS III from Invitrogen
Overview
Overview
Generate cDNA from isolated RNA. Generally, the procedure requires the addition of a short template that will bind to the RNA and then addition of an enzyme that will synthesize DNA using the RNA with the template attached. The template can be oligo(dT) (a string of TTTTTT), which will bind to the polyA tail of mRNA; random hexamers, which will bind anywhere the random sequences lie in the RNA; or specific primers, which are targeted to specific RNA. Currently we are using the Superscript III First-strand synthesis kit from Invitrogen. But also included in this protocol is the older Superscript II First-strand synthesis protocol. Read the Superscript III Platinum two-step qRT-PCR kit with SYBR Green instructions for more info.
Protocol
Protocol
- Perform all procedures on ice.
- Calculate number of samples
- Prepare master mix and master mix without RT enzymein 1.5 ml eppe.
- Incubate 65°C 5 min, return to ice for at least 1 min, spin down, and add the following\
- Mix by gently pipetting
- Incubate 50°C for 30 to 60 min
- Incubate 70°C for 15 min
- Add 1 µl of 2 U/µl RNase H to each tube
- Mix and incubate 37°C 20 min.