A quick look at my history of RT success to ID what started going wrong?
- not much smearing in early samples. Incorporation rate increases with total amount of mRNA added.
- Using beads to concentrate mRNA increases production.
- Using pure mRNA T7 further increases production
- Small samples have worse smears just after the high quality maxima integration (8 probes at 800 pmol P1, and 1 at nmol P2, 2 at 600 and 800 pmol P2).
- Correlates with switch to new P1. P3 was doing better than P1 for a little while after the switch.
- correlates with switch to murine RNase inhibitor from NEB (cheaper). Best runs were with RNasin Plus.
- This is interesting, RNasin plus advertises thermal stability up to 70C. Maybe this is the trick — NEB’s inhibitor says it should be used below 50C.
- correlates with lower IVT yields
- Same protocol essentially. Had higher maxima when we got high incorporation, but bringing maxima back up to high concentration doesn’t restore high incorporation.
- Nothing else changed really in protocol.
- some contamination of reagents.
- switch of RNase inhibitor.