Matt:LabNotes/2016-4-23: Difference between revisions
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==Protocol== | ==Protocol== | ||
===Day 1=== | ===Day 1=== | ||
#Prepare hydrogel mix | #Prepare hydrogel mix | ||
##Positive Control: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + | ##Positive Control: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4ul of PKP2 beads | ||
##5% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] | ##5% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] | ||
##5% PA Gel + 4% PFA: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4% PFA | ##5% PA Gel + 4% PFA: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4% PFA | ||
#Attach 3 Vectabond + Bind-silane treated coverslips | #Attach 3 ~120um thick adhesives to 3 Vectabond + Bind-silane treated coverslips | ||
#Add | #Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets | ||
#*Pipette away extra H2O leaving only dried beads | #*Pipette away extra H2O leaving only dried beads | ||
#Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip) | |||
#Inject ~ | |||
#Vacuum seal bag and then pump with Argon gas | #Vacuum seal bag and then pump with Argon gas | ||
#Let set at RT for 30min | #Let set at RT for 30min | ||
#Wash with nf-H2O twice | #Wash with nf-H2O twice | ||
#*5% PA Gel + 4% PFA failed to polymerize | |||
*Gels are sitting in 1X PBS at 4C | *Gels are sitting in 1X PBS at 4C | ||
#Prepare Ampligase mix on ice | #Prepare Ampligase mix on ice |
Revision as of 00:09, 29 April 2016
Test Diffusion of PA Gel Formulations
- Using PKP2 Beads as targets
- PKP2_controlPP
Dilute PKP2_controlPP
- Resuspended to 10uM with 450ul H2O
- Serial dilute to 10nM
- 10:1 dilution 3 times
Experiment Plan
- Goal: Test whether padlock probes and polymerases can diffuse through ~100um? polyacrylamide hydrogel to generate rolonies
- Dish1: Positive control: mix beads into hydrogel solution before polymerization
- This way some beads will be close to the surface, minimizing distance molecules need to diffuse
- Can't do it without hydrogel matrix because the beads and rolonies need some sort of substrate
- Dish2: 5% Polyacrylamide Gel (199:1 Acrylamide:Bis-acrylamide)
- Beads are underneath gel, being held in place by magnets
- Do padlock probe capture and then RCA to generate rolonies in hydrogel matrix
Protocol
Day 1
- Prepare hydrogel mix
- Positive Control: Standard hydrogel + 4ul of PKP2 beads
- 5% PA Gel: Standard hydrogel
- 5% PA Gel + 4% PFA: Standard hydrogel + 4% PFA
- Attach 3 ~120um thick adhesives to 3 Vectabond + Bind-silane treated coverslips
- Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
- Pipette away extra H2O leaving only dried beads
- Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
- Vacuum seal bag and then pump with Argon gas
- Let set at RT for 30min
- Wash with nf-H2O twice
- 5% PA Gel + 4% PFA failed to polymerize
- Gels are sitting in 1X PBS at 4C
- Prepare Ampligase mix on ice
- Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
DEPC-H2O | 70 |
Ampligase Buffer | 10 |
PKP2_controlPP 10nM | 10 |
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 ~24hr
Day 2
- Wash with 1X PBS once
- Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 70C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- Add RCA mix and incubate 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3
- Wash with 1X PBS twice
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris ph8.0 for 30min at RT
Imaging
Hybridize FISGA_Adpt Cy3
- Preheat 400ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
- Add 200ul to each dish and let sit for 10min at RT
- Wash twice with 2X SSC
- Dish 1
- Dish 2