Matt:LabNotes/2014-3-26
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Padlock Probe Concentration Test using Low Conc (1nM) Fixed Template
- Previously fixed 10uM template resulted in more targets than realistically would be in cells
- 24-well plate PGP1f Cells cultured w/o EGF
- Fixed 3/21/14 and RT 3/21/14
- Using wells A4, A5, D2, D3, D4, D5
- Marked 2 positions for each well (adapter clips on bottom&right)
- .maf file saved in 3-26-2014
- Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM
Fixing Template in cells
- Pre-heat 1nM MALAT1_Template in 2X SSC to 85C (6ul 100nM MALAT1_Template + 60ul 20X SSC + 534ul H2O)
- Add 100ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 100ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS)
- Incubate @RT for 1hr
- Wash with 1X PBS twice
- Add 50ul 1M Tris pH 8.0
- Incubate @ RT for 30min
- Wash with 1X PBS twice
Anneal Padlock Probes
- 1X Ampligase Buffer
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours
- Wrapped in parafilm and put in ziploc with wet towel
Negative Control 0pM (A4)
Component | Volume |
10X Ampligase Buffer | 10 |
5U/ul Ampligase | 10 |
H2O | 80 |
Total | 100 |
10pM (A5)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
50pM (D2)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 5 |
5U/ul Ampligase | 10 |
H2O | 75 |
Total | 100 |
100pM (D3)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
1nM (D4)
Component | Volume |
10X Ampligase Buffer | 10 |
10nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
10nM (D5)
Component | Volume |
10X Ampligase Buffer | 10 |
1uM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |