Matt:LabNotes/2016-8-3

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Revision as of 22:11, 3 August 2016 by >Mzcai (→‎To Sample 2 Only)
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DARTFISH on BA8 with and without PA Gel

  • Try 3 samples with polyacrylamide gel added at different points in protocol
  • Sample 1: No gel
  • Sample 2: Gel added after permeabilization
  • Sample 3: Gel added after reverse transcription
  • Used 200nM padlock probe (twice the normal concentration)
Component Volume
40% AB 199:1 Mix 6.25
10% BSA 1
1mM Acryd/Amine Linker 6.25
H2O 33.5
5% TEMED 1
5% APS 1
BS(PEG)9 1
Total 50

Protocol

Day 1

  1. Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Make 4% PFA in 1X PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out BA8 section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  9. Wash with nf-1X PBS three times and check for degradation

To Sample 2 Only

  • Skip these steps and go to Reverse Transcription Immediately for other 2 samples
  1. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  2. Add 50ul gel casting mix filtered and degassed
    • 5% gel with 125uM Acrydite-Amine linker and 5mM BS(PEG)9
  3. Seal in plastic bag and vacuum out air before filling with argon
  4. Let sit at RT for 30min
  5. Aspirate non-polymerized gel and wash once with 1X PBS
  6. Attach coverslip to bottom of petri dish
  7. Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS

Continue for All Samples

  1. Attach coverslip to bottom of petri dish
  2. 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 FISSEQ_RT primer 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C