AlanFung:LabNotes/Rolony/2014-3-5
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Secondary Rolony Generation for Padlock Probe Concentration Test[edit]
Samples[edit]
- 96 well plate containing NCIH-1975 cells fixed by Hosuk
- Location and save location of 12 position in 96 well plate for the experiment
- High Concentration (100pM)
- Low Concentration (1pM)
- Ultra Low Concentration (0.1pM)
Fixing Template in cells[edit]
- Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (60ul 100uM MALAT1_Template + 60ul 20X SSC + 480ul H2O)
- Add 50ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 50ul 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
Dilute 2nM ppMALAT1[edit]
- 100pM
- 1pM
- 0.1pM
Secondary RCA[edit]
- Aspirate each well 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 50ul to each well, 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 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
- Wash 1X PBS once
- Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
- Wash 1X PBS twice
- Add 50ul 1M Tris pH 8.0 @RT for 1hr
- Wash 1X PBS twice
Anneal Padlock Probes for 24 hours[edit]
- Wash twice w PBS
- 1X Ampligase Buffer (10ul of stock 10X)
- Add padlock probe to each well with their corresponding concentration (35ul)
- 0.5 U/ul Ampligase (5ul of stock 5U/ul)
- Incubate @ 45C for 24 hours
- Wrapped in parafilm and put in ziploc with wet towel
Group 1 100pM ppMALAT1[edit]
Group 1-100pM | Group 2-1pM | Group 3-0.1pM | ||||||||
Image | Well | Mark & Find | Image | Well | Mark & Find | Image | Well | Mark & Find | ||
A | C3 | 1 | E | D3 | 2 | I | C5 | 4 | ||
B | F8 | 15 | F | F5 | 14 | J | F6 | 7 | ||
C | F3 | 13 | G | C6 | 9 | K | C7 | 10 | ||
D | E6 | 8 | H | E7 | 11 | L | F7 | 12 |
3 | 4 | 5 | 6 | 7 | 8 | |
A | ||||||
B | ||||||
C | C3 | C5 | C6 | C7 | ||
D | D3 | |||||
E | E6 | E7 | ||||
F | F3 | F5 | F6 | F7 | F8 | |
G | ||||||
H |
File:Ppconcentration NCHI1975 Group1.jpg