Matt:LabNotes/2016-2-23: Difference between revisions
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>Mzcai m (→Day 2) |
>Mzcai m (→Day 4) |
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| Total||1000 | | Total||1000 | ||
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#Wash with 1X PBS twice | #Wash with 1X PBS twice and store at 4C overnight | ||
*REALIZED I USED THE WRONG RT PRIMER FOR FISSEQ, CAN'T DO RCA | *REALIZED I USED THE WRONG RT PRIMER FOR FISSEQ, CAN'T DO RCA | ||
*but can try padlock probe capture on circularized cDNA | |||
===Day 3=== | ===Day 3=== | ||
#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 | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| H2O|| | | 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|| | | Total||100 | ||
|} | |} | ||
#Add mix to sample and incubate for 30min at 37C | |||
===Day | #Move sample to 60C and oven slowly decreases to 55C and held for ~29hr | ||
===Day 4=== | |||
#Wash with 1X PBS once | #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 | |||
<!-- | |||
#To DARTFISH slide add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | #To DARTFISH slide add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
#*Preheated to 70C before adding | #*Preheated to 70C before adding |
Latest revision as of 01:17, 27 February 2016
DARTFISH on BA8 Sections with PA gel[edit]
- 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[edit]
Day 1[edit]
- 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[edit]
- 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
Day 3[edit]
- 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
Day 4[edit]
- 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