Revision as of 01:17, 27 February 2016 by >Mzcai
DARTFISH on BA8 Sections with PA gel[edit]
- 4% Formaldehyde is one month old and has been sitting at RT
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]
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- Used 4% PFA from 1/20/2016
- Take out worst BA8 sections (furthest front) 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
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 100ul gel casting mix filtered and degassed
- 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
- 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 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
- 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
- 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 ~15hr at 37C
- 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
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with nf-H2O twice
- Prepare FISSEQ mix on ice
- 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
|
- 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
- Wash with nfH2O twice
- 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
|
- Add mix to sample and incubate for 30min at 37C
- Move sample to 60C and oven slowly decreases to 55C and held for ~29hr
- 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