Matt:LabNotes/2016-4-28: Difference between revisions
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>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...") |
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=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 | ||
==Experiment Plan== | ==Experiment Plan== | ||
*Goal: Test whether padlock probes | *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== | ==Protocol== |
Revision as of 03:48, 29 April 2016
Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes
- Using PKP2 Beads as targets
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)
- Previously PKP2_controlPP worked but in that experiment:
Protocol
Day 1
- Prepare hydrogel mix
- 5% PA Gel: Standard hydrogel
- Attach ~120um thick adhesive to Vectabond + Bind-silane treated coverslip
- Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
- Pipette away extra H2O leaving only dried beads
- Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
- Vacuum seal bag and then pump with Argon gas
- Let set at RT for 30min
- Attach to bottom of culture dish
- 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
Component | Volume |
DEPC-H2O | 52 |
Ampligase Buffer | 10 |
Batch 15 368nM | 28 |
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