Matt:LabNotes/2016-4-11: Difference between revisions
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>Mzcai m (→Dish 2: 10% PA) |
>Mzcai |
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*Beads well dispersed among many z layers | *Beads well dispersed among many z layers | ||
**No z-slice has them all in focus | **No z-slice has them all in focus | ||
[[]] | |||
====Hybridize FISGA_Adpt Cy3==== | ====Hybridize FISGA_Adpt Cy3==== | ||
#Preheat 600ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C | #Preheat 600ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C | ||
#Add 200ul to each dish and let sit for 10min at RT | #Add 200ul to each dish and let sit for 10min at RT | ||
#Wash twice with 2X SSC | #Wash twice with 2X SSC | ||
*Position 1 | |||
[[]] | |||
*Position 2 | |||
**See some spots but they overlap with bead position (and not the beads closest to the top surface) | |||
[[File:Dish1_PosCtrl_FISGA_Adpt_Pos2_z07_ch01.jpg|450px]][[File:MAX_Dish1_PosCtrl_FISGA_Adpt_Pos2.jpg|450px]] | |||
====Strip==== | ====Strip==== | ||
#Preheat 80% formamide 2X SSC to 75C | #Preheat 80% formamide 2X SSC to 75C |
Revision as of 20:51, 22 April 2016
Test Diffusion of PA Gel Formulations
- Using PKP2 Beads as targets
- CA12kNov2014 V4 Padlock Probes
Experiment Plan
- Goal: Test whether padlock probes and polymerases can diffuse through ~400um 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: 10% Polyacrylamide Gel (199:1 Acrylamide:Bis-acrylamide)
- Beads are underneath gel, being held in place by magnets
- Dish3: 10% Polyacrylamide (199:1) + 4% PFA Gel
- 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 + 2ul of PKP2 beads
- 10% PA Gel: Standard hydrogel
- 10% PA Gel + 4% PFA: Standard hydrogel + 4% PFA
- Attach 3 Vectabond + Bind-silane treated coverslips to 3 culture dishes
- Add 3ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
- Pipette away extra H2O leaving only dried beads
- File:20160411 magnetbeads culturedish.jpg
- Inject ~70ul of 200ul hydrogel mix into each dish (underneath the plastic insert)
- Vacuum seal bag and then pump with Argon gas
- Let set at RT for 30min
- Wash with nf-H2O twice
- Prepare Ampligase mix on ice
- Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme
- Combine B11 and B12: 100ul at ~200nM
- B12 looked to have some very fine PA gel still in there (not filtered enough?)
Component | Volume |
DEPC-H2O | 47 |
Ampligase Buffer | 10 |
Batch 11 & 12 Padlock Probes ~200nM]] | 33 |
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
- Dish1 showed signs of leaking but the gel was not dried out and there was still some liquid on top
- Later confirmed to definitely be leaking
- 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
Dish 1: Positive Control
- Beads well dispersed among many z layers
- No z-slice has them all in focus
[[]]
Hybridize FISGA_Adpt Cy3
- Preheat 600ul 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
- Position 1
[[]]
- Position 2
- See some spots but they overlap with bead position (and not the beads closest to the top surface)
File:Dish1 PosCtrl FISGA Adpt Pos2 z07 ch01.jpgFile:MAX Dish1 PosCtrl FISGA Adpt Pos2.jpg
Strip
- Preheat 80% formamide 2X SSC to 75C
- Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT
- Wash twice with 1X PBS
- Add 500ul 2x SSC to image
Hybridize Cycle5 Cy3 & Cy5
- Only Cy5 should show signal
Dish 2: 10% PA
- Beads are clearly in focus in single z-plane
- High density where magnet was
- Hybridized FISGA_Adpt_Cy3 but only a few fluorescent spots, not sure if rolonies or beads...
- Turned up brightness in ImageJ and can see dim beads
- However if the few bright spots are rolonies I expected to see more
Dish 3: 10% PA 4% PFA
- Beads are clearly in focus in single z-plane
- High density where magnet was
File:Dish3 10%PA 4%PFA z07 ch01.jpg
- Hybridized FISGA_Adpt_Cy3 but No Fluorescence!!
- Turned up brightness in ImageJ and only signal is from beads
File:MAX Dish3 10%PA 4%PFA.jpg
Order PKP2 Padlock Probe from IDT
- To run these tests without wasting time/$/effort on padlock probe production from CA12k_Nov2014 V4
- Order single padlock probe from IDT
PKP2 PKP2_3 chr12:32802524-32802525 GAGATGGCTGTCTTTTTCACACTTGG 63 GTCACCAACATGCAGCATCTTTC 63 2.719207 W GAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC B02203
- PKP2_controlPP: /5Phos/CCAAGTGTGAAAAAGACAGCCATCTCCTTCAGCTTCCCGATATCCGACGGTAGTGTCGAAAGATGCTGCATGTTGGTGAC