Matt:LabNotes/2016-4-23

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Revision as of 20:47, 23 April 2016 by >Mzcai (Created page with "=Test Diffusion of PA Gel Formulations= *Using PKP2 Beads as targets *PKP2_controlPP ==Dilute PKP2_controlPP== *Resuspended to 10uM with 450ul H2O...")
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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