Hosuk:LabNotes/2014-9-30
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ppCOL1A1 + Suppressor test
- Sample : 4 dishes PGP1F, 1st Rolonies made at 2014-07-17
- Check 1st rolonies of all 5 dishes
- S1, S4 --> ppCOL1A1 + suppCOL1A1
- S2, S5 --> ppCOL1A1 only
- Add suppressor 10 times more than ppCOL1A1
Procedure
- ppCOL1A1 + supp_COL1A1 : S1, S4
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
100uM ppCOL1A1 | 2 |
100uM Supp_COL1A1 | 20 |
H2O | 58 |
Total | 100 |
- ppCOL1A1 : S2, S5
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
100uM ppCOL1A1 | 2 |
100uM Supp_COL1A1 | 0 |
H2O | 78 |
Total | 100 |
- Incubate at 37C for 30min
- Incubate at 60C for 24hrs
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
- Wash with 2X SSC twice
- Add 200ul RCA Mix and incubate at 30C for 16hrs
- H2O : 172uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (1000 U/µl) : 0.5uL
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
- Wash with 1X PBS twice
- Add 200ul 1M Tris pH 8.0 at RT for 30min
- Wash with 1X PBS twice and store in PBS