Objective: Convert RNA to cDNA with Sam at the Friedman lab used following reverse transcription protocol
Reverse Transcription (Promega M-MLV: Cat#M1701; ) [Cost per sample ~$1.50]
A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.
1. Use as much RNA as possible (up to 1ug); max volume of RNA = 17.75uL. Generally, identify the RNA sample with the lowest concentration and multiply by 17.75uL. Use this quantity (ug) of RNA for each and every sample.
2. Transfer calculated volume(s) of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
3. Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.
4. Heat samples at 70C for 5 min in thermocycler.
5. Place samples on ice IMMEDIATELY.
6. Make Master Mix:
5 uL 5x Buffer (M-MLV RT Buffer)
1.25 uL 10mM dNTPs (Promega Cat#U1511)
0.5 uL M-MLV RT per ug of RNA
Used 4uL of RNA (
1ul of rna from each of the original sea pen sample added to a single sample combining rna sp1-1 sp1-2 sp2-1 sp2-2)
7. Mix well.
8. Add 6.75uL of master mix to each reaction.
9. Mix well, but do not vortex.
11.Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.
12.Heat inactivate @ 95C for 3 min.
14.Store @ -20C.
Sucessfully converted RNA to cDNA next step is to replicate using PCR and verify on a gel.