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
    1. Extract (qiagen)
    2. OD260 to get concentration (can figure out how much should be rRNA and hence what the quantitative numbers are
    3. 100-500ng (250ng optimal, try going down to 50ng to keep in linear range of detection?.
    4. Agarose gel, 1% agarose, 2% formamide
    5. Loading buffer 50% formamide, 10mM Tris, 10% glycerol, whatever dye I want. TAE fine for running gel
    6. Run gle
    7. Transfer overnight using turboblotter
      • Bryan offered to help us with this first time through.
    8. Crosslinking with UV for 5 min
    9. Go straight to labeling and detection kit (Bryan has some reagents left over which he thinks should be fine)
    10. Use 42C incubator with roller to bake 50ml conical tube during the protocol.
    11. Image via fluorescence (better quantification, more linear?) or chemiluminescence (better sensitivity)
    12. 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
    1. Our imager should be fine, or use film!
  • Relative vs "quantitative"
    1. Bryan mainly does relative.


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