Matt:LabNotes/2016-2-23

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DARTFISH on BA8 Sections with PA gel[edit]

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[edit]

Day 1[edit]

  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
    • FORGOT TO DO THIS SO GEL WON'T BE FLAT ON TOP
  9. Seal in plastic bag and vacuum out air before filling with argon
  10. Let sit at RT for 30min
  11. Aspirate non-polymerized gel and wash once with cold 1X SSPE
    • Mostly did not polymerize
    • Only a ~1cm diameter circle of gel polymerized in center
    • I will continue with FISSEQ protocol to see whether can generate as many rolonies in gel area as non-gel area
  12. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  13. Wash with cold nf-H2O three times and check for degradation
  14. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  15. Wash with nf-1X PBS three times and check for degradation
  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 ~15hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. Prepare FISSEQ mix on ice
  9. Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component Volume
DEPC-H2O 128
CircLigase Buffer 10X 20
MnCl2 50mM 10
Betaine 5M 40
CircLigase II (100U/ul) 2
Total 1000
  1. Wash with 1X PBS twice and store at 4C overnight
  • REALIZED I USED THE WRONG RT PRIMER FOR FISSEQ, CAN'T DO RCA
  • but can try padlock probe capture on circularized cDNA

Day 3[edit]

  1. Wash with nfH2O twice
  2. Prepare Ampligase mix on ice
    • Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme
  • B13: 305nM
    • This batch of padlock probes was PCR'd an extra 3 cycles during production PCR
Component Volume
DEPC-H2O 18.43
Ampligase Buffer 10
Erin's Batch 13 2/12/2016 Padlock Probes 305nM]] 33
100nM suppv2 Oligos 28.57
Ampligase 10
Total 100
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~29hr

Day 4[edit]

  1. Wash with 1X PBS once
  • LEAK! no idea why it started leaking when it has been fine until now...
  • Can't do RCA, just put in 4C storage