Matt:LabNotes/2016-7-5: Difference between revisions

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#Attach coverslip to bottom of petri dish
#Attach coverslip to bottom of petri dish
#Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
#Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
#*No signs of leaking
#Prepare Reverse Transcription Mix '''on ice''' and add
#Prepare Reverse Transcription Mix '''on ice''' and add
{| {{table}}
{| {{table}}
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| Total||200
| Total||200
|}
|}
#Incubate 10min at 4C and then ~18hr at 37C<!--
#Incubate 10min at 4C and then ~18hr at 37C
===Day 2===
===Day 2===
*There was almost no solution left on coverslip though there was around edges of dish
*RT solution leaked under the dish and the gel/tissue were essentially dry
**Was it bumped, tilted, or moved by someone?
*Gel does not look significantly swollen though
*Gel swollen and wrinkled as expected
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
#Wash with 1X PBS twice
#Wash with 1X PBS twice
*Leak is too bad, unusable
<!--
#Add 1M Tris pH 8.0 and incubate 30min at RT
#Add 1M Tris pH 8.0 and incubate 30min at RT
#Wash with 1X PBS twice<!--
#Wash with 1X PBS twice
#RNA Removal
#RNA Removal
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C

Latest revision as of 01:25, 13 July 2016

DARTFISH on BA8 sections with PA gel + Acrydite/BS(PEG)9[edit]

  • Picked up 10 fresh BA8 sections from Yun placed on Vectabond + Bind-silane treated coverslips
Component Volume
40% AB 199:1 Mix 12.5
10% BSA 2
Acryd/Amine Linker 50
H2O 29.5
5% TEMED 2
5% APS 2
BS(PEG)9 2
Total 100

Protocol[edit]

Day 1[edit]

  1. Prepare two plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Make fresh 4% PFA in 1X PBS
  3. Take out BA8 section 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. 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
    • Lots of tissue degraded off glass, maybe Vectabond + Bind-silane is not enough Vectabond
    • Repeat one more time and hope this was a fluke
    • Second time around there was tissue remaining
  10. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  11. Add 100ul gel casting mix filtered and degassed
    • Capillary action pulls gel mix between glass surfaces but there were a few small (~1mm diameter) air bubbles
    • 5% gel with 500uM Acrydite-Amine linker and 5mM BS(PEG)9
  12. Seal in plastic bag and vacuum out air before filling with argon
  13. Let sit at RT for 30min
  14. Aspirate non-polymerized gel and wash once with 1X PBS
  15. Attach coverslip to bottom of petri dish
  16. Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
    • No signs of leaking
  17. 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

Day 2[edit]

  • RT solution leaked under the dish and the gel/tissue were essentially dry
  • Gel does not look significantly swollen though
  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


  • Leak is too bad, unusable