Matt:LabNotes/2016-3-14

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Revision as of 00:30, 15 March 2016 by >Mzcai (→‎Day 1)
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DARTFISH on BA8 Sections with PA gel

Protocol

Day 1

  1. Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Used 4% PFA from 3/8/2016
  3. Take out second best remaining BA8 sections (4th from back) from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times and check for degradation
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nf-1X PBS three times and check for degradation
  11. Add 75ul gel casting mix filtered and degassed
    • 4% PFA, 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED
  12. Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height
  13. Seal in plastic bag and vacuum out air before filling with argon
  14. Let sit at RT for 30min
  15. Aspirate non-polymerized gel and wash once with cold 1X SSPE
    • Edges did not polymerize but the grey matter is fully covered
  16. Prepare Reverse Transcription Mix on ice and add
Components Volume
H2O 159
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C