Matt:LabNotes/2016-4-23: Difference between revisions
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>Mzcai m (→Protocol) |
>Mzcai m (→Day 2) |
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===Day 3=== | ===Day 3=== | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT | #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 1M Tris ph8.0 for 30min at RT--> | #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 | |||
<!-- | |||
====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 | |||
*Bumped dish, lost position but found again | |||
*Position 1 (Cy3) | |||
**Not good sign, signal is still there... even though it looks like less | |||
[[File:MAX_Dish1_PosCtrl_Stripped_Cy3_Pos1.jpg|450px]] | |||
*Position 1 (Cy5) | |||
[[File:MAX_Dish1_PosCtrl_Stripped_Cy5_Pos1.jpg|450px]] | |||
====Hybridize Cycle5 Cy3 & Cy5==== | |||
*Only Cy5 should show signal | |||
*Position 1 (Cy3) | |||
[[File:MAX_Dish1_PosCtrl_Cycle5_Cy3_Pos1.jpg|450px]] | |||
*Position 1 (Cy5) | |||
**Good there's signal when there wasn't before! | |||
[[File:MAX_Dish1_PosCtrl_Cycle5_Cy5_Pos1.jpg|450px]] | |||
===Dish 2: 10% PA=== | |||
*Beads are clearly in focus in single z-plane | |||
**High density where magnet was | |||
[[File:Dish2_10%25PA_z07_ch01.jpg|450px]] | |||
*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 | |||
[[File:MAX_Dish2_10%25PA.jpg|450px]] | |||
===Dish 3: 10% PA 4% PFA=== | |||
*Beads are clearly in focus in single z-plane | |||
**High density where magnet was | |||
[[File:Dish3_10%25PA_4%25PFA_z07_ch01.jpg|450px]] | |||
*Hybridized FISGA_Adpt_Cy3 but No Fluorescence!! | |||
**Turned up brightness in ImageJ and only signal is from beads | |||
[[File:MAX_Dish3_10%25PA_4%25PFA.jpg|450px]]--> |
Revision as of 19:24, 25 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
- 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