Matt:LabNotes/2016-4-11: Difference between revisions
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>Mzcai mNo edit summary |
>Mzcai m (→Day 2) |
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| Total||200 | | Total||200 | ||
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===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 |
Revision as of 18:47, 13 April 2016
- 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
- Combine B11 and B12: 100ul at ~200nM
- B12 looked to have some very fine PA gel still in there (not filtered enough?)
Component | Volume |
DEPC-H2O | 47 |
Ampligase Buffer | 10 |
Batch 11 & 12 Padlock Probes ~200nM]] | 33 |
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