Daniel:Notebook/RNAFISH/2014-12-15

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Revision as of 21:09, 15 December 2014 by >Djacobse (Created page with "=Cell Line RNA FISH 2= Back to Calendar Since I have two more samples and not enough time to really do much else, I'm going to retry the cell lin...")
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Cell Line RNA FISH 2

Back to Calendar

Since I have two more samples and not enough time to really do much else, I'm going to retry the cell line FISH.

Hybridization

  1. Permeabilize by leaving at room temperature for 30 minutes in 2X SSCT+50% formamide
  2. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  3. Dry in oven at 50-65C.
  4. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  5. Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
  6. Drop temp to 65C
  7. Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
  8. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  9. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  10. Hybridize overnight appropriate temperature
    1. VARIABLE--shorter probes may want much lower temp; Hybridization temp may need to be determined empirically (=many trials…….)
    2. important to keep slides in a humid environment
  • Prehybridization solution:
    • 50% deionized formamide
    • 50% 2xSSC pH 7.0
    • Add dextran sulfate to 1%
  • Hybridization solution (Arjun Raj)
    • 2X SSC
    • 10% Formamide
    • 10% Dextran Sulfate
    • 0.02% RNAse-free BSA
    • 50 μg E.coli tRNA