Matt:LabNotes/2014-10-6
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Testing Agi26k0gap + suppressor Oligos & Agi26k20gap[edit]
Sample[edit]
- Agi26k0gap Control: PGP1F Fix 5.19.14 S2
- Agi26k0gap + suppressors: PGP1F Fix 5.19.14 S3
- Agi26k20gap: Fix 10.1.14
Procedure[edit]
- Agi26k0gap Control
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
Agi26k0gap | 35 |
H2O | 45 |
Total | 100 |
- Agi26k0gap + suppressors
- Combined 1ul of each 100uM 6 suppressor oligos in 1ml total to make 600nM oligo mix (100nM each suppressor oligo)
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
Agi26k0gap | 35 |
600 nM (100nM each) supp_Mix | 5 |
H2O | 40 |
Total | 100 |
- Agi26k20gap
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
Agi26k20gap | 70 |
25mM dNTP | 1 |
10 U/ul KlenTaq | 2 |
H2O | 7 |
Total | 100 |
- Incubate at 37C for 30 min
- Incubate at 60C for 24 hrs
- 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 : 173uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (1000 U/µl) : 1uL
- 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