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 20 hours
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 |
Secondary RCA
- Aspirate but don't wash
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 100ul and incubate at 45C for 15min.
- Aspirate, and wash using 2x SSC twice
- Aspirate, and wash using 0.1x SSC twice
- Prepare the rolling circle amplification reaction mixtures on ice.
- 513 ul ddH2O,
- 60 ul 10x Phi29 buffer,
- 6 ul 25 mM dNTP,
- 12 ul 2 mM aminoallyl dUTP
- 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
- 600ul Total Volume
- Add 100ul each well and incubate 20hrs at 30C. (Put in at 1:30pm 3/27/14)
- Wash 1X PBS once
- Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr
- Wash 1X PBS twice
- Add 100ul 1M Tris pH 8.0 @RT for 30min
- Wash 1X PBS twice
Detection
- Made 600ul of 1uM dcProbe2-Cy3 (594ul 2XSSC & 30%formmaide + 6ul 100uM dcProbe2-Cy3)
- Pre-heated @75C for 5 min and then added 100ul to each well
- Let sit @RT for 5 min
- Washed twice with 2X SSC
- Saved in 3-28-2014