Matt:LabNotes/2016-7-5
Jump to navigation
Jump to search
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
- Gel mix and protocol is same as this with BS(PEG)9
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]
- Prepare two plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Make fresh 4% PFA in 1X PBS
- Take out BA8 section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- 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
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- 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
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with 1X PBS
- Attach coverslip to bottom of petri dish
- 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
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 |
- 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
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Leak is too bad, unusable