User:Barry Canton/Notebook/T7 RNAP transcription of rRNA/2008/06/20

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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.

Timeline

 * 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.


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