Matt:LabNotes/2016-1-20

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Revision as of 22:51, 20 January 2016 by >Mzcai (Created page with "=DARTFISH on BA8 Sections= *[Matt:LabNotes/2015-10-29|Last time imaged with 20X objective didn't decode enough rolonies] **90 sec 0.01% pepsin incubation worked well *Picked ...")
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DARTFISH on BA8 Sections

  • [Matt:LabNotes/2015-10-29|Last time imaged with 20X objective didn't decode enough rolonies]
    • 90 sec 0.01% pepsin incubation worked well
  • Picked up 9 sections from Yun today
    • He said many of the sections had "shattering" near the superficial layers of the thicker end (The curved part of 'P')
    • The best sections are in the back of the box and the worst are in the front
      • The very worst had tissue wiped off one side of coverslip and then better section stuck to the other side

Protocol

Day 1

  1. Prepare 2 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Prepare fresh 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
  3. Take out 2 best BA8 sections (2 furthest 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
    • Slightly more stripes in gray matter
  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. 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