I am basically trying to determine the sequences of a mature protein in a variety of lines.
So for this I have;
- extracted RNA,
- treated with DNase
- performed cDNA synthesis.
- PCR with gene-specific primers
- column clean up of excised band
- ligate into PGEM-T easy vector
- heat-shock into competent cells
- grow up in SOC media
- spread on 2xYT Plate
- Colony PCR to check insert
- grow up positive colonies in 2xYT
- use Wizard Miniprep purification system
- Sequence clones.
am I missing anything here? The problem is, somebody is 'teaching' me and they really aren't very good at explaining or answering questions on why we do each step, and what each reagent does etc. I have got to grips to this basic method and understand everything fine etc, but then people keep shooting questions at me about Poly A tailing and mRNa and so on.
When would you Poly A tail and why? And is what I have been doing correct?
Thanks so much for any help - I've been searching the internet and books for hours and hours and just can't quite figure it out.