Matt:LabNotes/2016-4-28: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
(Created page with "=Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes= *Using PKP2 Beads as targets *PKP2_controlPP ==Dilute PKP2_controlPP== *Resuspend...")
 
>Mzcai
Line 1: Line 1:
=Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes=
=Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes=
*Using [[Matt:LabNotes/2016-3-31|PKP2 Beads]] as targets
*Using [[Matt:LabNotes/2016-3-31|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==
==Experiment Plan==
*Goal: Test whether padlock probes and polymerases can diffuse through ~100um? polyacrylamide hydrogel to generate rolonies
*Goal: Test whether the longer (150bp) padlock probes can diffuse through ~120um polyacrylamide hydrogel to generate rolonies
*Dish1: Positive control: mix beads into hydrogel solution before polymerization
**Previously PKP2_controlPP worked but in that experiment:
**This way some beads will be close to the surface, minimizing distance molecules need to diffuse
***The concentration was 33X higher than in actual DARTFISH probeset (1nM vs 30pM)
**Can't do it without hydrogel matrix because the beads and rolonies need some sort of substrate
***PKP2_controlPP is half the length (~70bp vs 150bp)
*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==
==Protocol==

Revision as of 03:48, 29 April 2016

Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes

Experiment Plan

  • Goal: Test whether the longer (150bp) padlock probes can diffuse through ~120um polyacrylamide hydrogel to generate rolonies
    • Previously PKP2_controlPP worked but in that experiment:
      • The concentration was 33X higher than in actual DARTFISH probeset (1nM vs 30pM)
      • PKP2_controlPP is half the length (~70bp vs 150bp)

Protocol

Day 1

  1. Prepare hydrogel mix
    1. 5% PA Gel: Standard hydrogel
  2. Attach ~120um thick adhesive to Vectabond + Bind-silane treated coverslip
  3. Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
    • Pipette away extra H2O leaving only dried beads
  4. Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
  5. Vacuum seal bag and then pump with Argon gas
  6. Let set at RT for 30min
  7. Attach to bottom of culture dish
  8. Wash with nf-H2O twice
  9. 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 52
Ampligase Buffer 10
Batch 15 368nM 28
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