Matt:LabNotes/2016-11-16: Difference between revisions
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=DARTFISH on | =DARTFISH on 5342 Occipital Cortex with SplintR in PA Gel= | ||
*Try 1 sample with polyacrylamide gel added [[Matt:LabNotes/2016-8-3|after permeabilization]] | *Try 1 sample with polyacrylamide gel added [[Matt:LabNotes/2016-8-3|after permeabilization]] | ||
*Used 100nM padlock probe | *Used 100nM padlock probe | ||
*Sample: GTEx Patient 5342 | *Sample: GTEx Patient 5342 Occipital Cortex | ||
*5% Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_Mix|this with Acryloyl-X]] | *5% Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_Mix|this with Acryloyl-X]] | ||
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#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O | #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O | ||
#Wash with nf-1X PBS three times '''and check for degradation''' | #Wash with nf-1X PBS three times '''and check for degradation''' | ||
#*First try: ALL the tissue sloughed off by the end of 3rd wash | |||
#*Second try: Decreased pepsin time to 60sec and only the areas circled in red in image below sloughed off so continued | |||
#**Polymerization of gel was successful but when trying to remove coverslip from glass slide the coverslip cracked | |||
#*Third try: With same pepsin time of 60sec all tissue sloughed off | |||
#*Fourth try: Skipped pepsin step but then dropped coverslip face down on bench... SHIT! | |||
[[File:IMG_0297.jpg|350px]] | |||
#Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue | #Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue | ||
#*Secure in plastic jig | #*Secure in plastic jig | ||
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| Total||100 | | Total||100 | ||
|} | |} | ||
#Move sample to 60C and | *Padlock probes are mixed with SplintR Buffer but since they weren't used put in 4C box labeled "Matt" | ||
**May need to spike in ATP next time it is used | |||
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#Move sample to 60C oven and slowly decreases to 55C and held for ~24hr | |||
===Day 2=== | ===Day 2=== | ||
#Wash with 1X PBS twice preheated to 55C | #Wash with 1X PBS twice preheated to 55C |
Latest revision as of 03:10, 19 November 2016
DARTFISH on 5342 Occipital Cortex with SplintR in PA Gel[edit]
- Try 1 sample with polyacrylamide gel added after permeabilization
- Used 100nM padlock probe
- Sample: GTEx Patient 5342 Occipital Cortex
- 5% Gel mix and protocol is same as this with Acryloyl-X
Component | Volume |
40% AB 199:1 Mix | 6.25 |
10% BSA | 1 |
10mg/ml Acryloyl-X, SE in DMSO | 1 |
H2O | 39.75 |
5% TEMED | 1 |
5% APS | 1 |
Total | 50 |
Protocol[edit]
Day 1[edit]
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Made 4% PFA in 1X PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
- Take out brain section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish 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
- First try: ALL the tissue sloughed off by the end of 3rd wash
- Second try: Decreased pepsin time to 60sec and only the areas circled in red in image below sloughed off so continued
- Polymerization of gel was successful but when trying to remove coverslip from glass slide the coverslip cracked
- Third try: With same pepsin time of 60sec all tissue sloughed off
- Fourth try: Skipped pepsin step but then dropped coverslip face down on bench... SHIT!
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- 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 1X PBS
- Prepare SplintR Mix
- Preheat Padlock Probes then snap cool
Component | Volume |
10X SplintR Buffer | 10 |
CA12kOct2016_V4 160nM | 60 |
H2O | 30 |
Total | 100 |
- Padlock probes are mixed with SplintR Buffer but since they weren't used put in 4C box labeled "Matt"
- May need to spike in ATP next time it is used