Things to do
- Purchase reagents for RNA extraction via Sean's protocol
- Purchase reagents for Northern blotting according to Brian's protocol
- Grow cells
- Lyse (denaturing lysis is fine)
- How do we store lysates
- Design and order probes
- Design and order a standard to compare hybridization efficiency.
- Do test Northerns to check primer.
- Decide if we should try to get quantitative measures of rRNA levels or simply pursue measurements relative to wild-type rRNA levels.
- Order reagents today
- Do extractions Tuesday/Wednesday
- Do trial Northern Thursday
- Try polysome stuff the following week
Questions for Bryan
- Any problems with RNAse when doing the native lysis?
- Hasn't had any, thinks the Qiagen kit helps keep degradation down and also that the rRNAs are pretty stable
- Protocol for Northerns
- Extract (qiagen)
- OD260 to get concentration (can figure out how much should be rRNA and hence what the quantitative numbers are
- 100-500ng (250ng optimal, try going down to 50ng to keep in linear range of detection?.
- Agarose gel, 1% agarose, 2% formamide
- Loading buffer 50% formamide, 10mM Tris, 10% glycerol, whatever dye I want. TAE fine for running gel
- Run gle
- Transfer overnight using turboblotter
- Bryan offered to help us with this first time through.
- Crosslinking with UV for 5 min
- Go straight to labeling and detection kit (Bryan has some reagents left over which he thinks should be fine)
- Use 42C incubator with roller to bake 50ml conical tube during the protocol.
- Image via fluorescence (better quantification, more linear?) or chemiluminescence (better sensitivity)
- Some tweaks -
- more/less RNA
- more/less probe
- change oven temp? 42 should be fine
- Annealing temp. for hybridization, may need to go pretty high above Tm to make it specific
- Equipment needed to image gel
- Our imager should be fine, or use film!
- Relative vs "quantitative"
- Bryan mainly does relative.