Matt:LabNotes/2016-2-23

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DARTFISH on BA8 Sections with PA gel

3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5."

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 1/20/2016
  3. Take out worst BA8 sections (furthest front) 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 100ul gel casting mix filtered and degassed
  8. Cover with plastic coverslips cut and glued together such that it leaves 0.5mm space for gel height
  9. Seal in plastic bag and vacuum out air before filling with argon
  10. Let sit at RT for 30min
  11. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  12. Wash with cold nf-H2O three times and check for degradation
  13. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  14. Wash with nf-1X PBS three times and check for degradation
  15. 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