Matt:LabNotes/2016-2-23: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| DEPC-H2O||640
| DEPC-H2O||128
|-
|-
| CircLigase Buffer 10X||100
| CircLigase Buffer 10X||20
|-
|-
| MnCl2 50mM||50
| MnCl2 50mM||10
|-
|-
| Betaine 5M|| 200
| Betaine 5M|| 40
|-
|-
| CircLigase II (100U/ul)||10
| CircLigase II (100U/ul)||2
|-
|-
| Total||1000
| Total||1000
|}
|}
#Wash with 1X PBS twice
#Wash with 1X PBS twice
#Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
#Add 200ul 0.5uM FISSEQ_RCA (1ul 100uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
#Wash with 2X SSC + 30% formamide for 10min at 60C
#Wash with 2X SSC + 30% formamide for 10min at 60C
#wash with 2X SSC, 1X SSC, 1X PBS once each
#wash with 2X SSC, 1X SSC, 1X PBS once each
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| Total||200
| Total||200
|}
|}
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===Day 3===
===Day 3===

Revision as of 23:42, 24 February 2016

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
    • 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

  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
  2. Add 200ul 0.5uM FISSEQ_RCA (1ul 100uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  3. Wash with 2X SSC + 30% formamide for 10min at 60C
  4. wash with 2X SSC, 1X SSC, 1X PBS once each
  5. Add 200ul RCA mix and incubate for 15hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200