Daniel:Notebook/RNAFISH/2015-3-5

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Revision as of 18:57, 5 March 2015 by >Djacobse (→‎Hybridization)
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Linnarsson Lab Protocol 2 (Started 3/4/2015)

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Dye Coupling (Continued; Ethanol Precipitation)

  1. Remove from -80C
  2. Centrifuge at 4 C at 12000 rpm for 30 minutes
  3. Remove supernatant and add 700 uL chilled 75% EtOH
  4. Centrifuge at minutes at 4 C for 10 minutes at 12000 rpm
  5. Remove supernatant and dry the pellet in the hood
  6. Resuspend pellet in 7.5 uL TE buffer
  7. Store DNA at 4C for immediate use

Nanodrop Results

  ng/uL ssDNA pmol/uL dye ng/uL ssDNA pmol/uL dye dye/probe bp:dye ratio EtOH Yield (%) DNA EtOH Yield Dye (%) Total Yield DNA (%) uM Labeled DNA
FOXP2-488 23.5 6.7 230.3 40.0 1.1 17 73.50 44.78 34.55 34.9
GAD1-546 30.8 14.7 292.1 53.3 1.2 17 71.13 27.19 43.82 44.3
GAD1-594 52.2 31.6 351.7 66.1 1.2 16 50.53 15.69 52.76 53.3
FOXP2-647 28.3 27.7 311.2 54.0 1.1 17 82.47 14.62 46.68 47.2

Some of my best labeling results yet. The stock solutions are all almost twice as high as Simone's protocol calls for.

Hybridization

Sample Matrix

  uL FOXP2-488 (pmol) uL GAD1-546 (pmol) uL GAD1-594 (pmol) uL FOXP2-647 (pmol)
Sample A 0 5 (220) 0 5 (235)
Sample B 5 (175) 0 5 (265) 0

Protocol

  1. Prepare fresh paraformaldehyde 4% and keep at RT
  2. Fix sections with 4% paraformaldehyde for 15 minutes at RT
  3. Rinse sections twice with RT PBS
  4. Permeabilization-may use:
    1. Ethanol (70% EtOH, >90 minute incuabation at 4C)
  5. Rinse sections twice with RT PBS
  6. Prepare the wash buffer and let it sit at RT
  7. Warm hybridization buffer and warm it up to 37C
  8. Prepare hybridization reaction:
    1. hybridization buffer-100 uL; probe 2-10 uL
  9. Incubate o/n - 24 hours at 37C depending on tissue/thickness/etc.