Matt:LabNotes/2016-4-23: Difference between revisions

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===Day 3===
===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
#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

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
  1. 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
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 2

  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. 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

  1. Wash with 1X PBS twice
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris ph8.0 for 30min at RT

Imaging

Hybridize FISGA_Adpt Cy3

  1. Preheat 400ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
  2. Add 200ul to each dish and let sit for 10min at RT
  3. Wash twice with 2X SSC
  • Dish 1
  • Dish 2