Matt:LabNotes/2016-2-23: Difference between revisions
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===Day 3=== | |||
#Add 200ul 0.5uM FISSEQ_RCA (1ul 100uM FISSEQ_RCA + 199ul 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 |
Revision as of 02:54, 25 February 2016
DARTFISH on BA8 Sections with PA gel
- Used worst section from 1/19/16 BA8 patient 1568 from Yun
- Gel mix and protocol is same as this successful experiment
- 4% Formaldehyde is one month old and has been sitting at RT
- From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf>
- "We recommend that 10% buffered formalin solutions be used no longer than
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
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- 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
Day 2
- 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
- REALIZED I USED THE WRONG RT PRIMER FOR FISSEQ, CAN'T DO RCA