AlanFung:LabNotes/Rolony/2014-3-5
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Secondary Rolony Generation for Padlock Probe Concentration Test
Samples
- 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)
Fixing Template in cells
- Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (10ul 100uM MALAT1_Template + 10ul 20X SSC + 80ul H2O)
- Add 50ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Wash with 1X PBS once in C10
- Add 50ul BS(PEG)9 mix (2ul BS(PEG)9 + 98ul 1X PBS)
- Incubate @RT for 1hr
- Wash with 1X PBS twice
- Add 50ul 1M Tris pH 8.0
- Incubate @ RT for 30min
Anneal Padlock Probes for 24 hours
- 1X Ampligase Buffer
- Various padlock probe concentration
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours
- Wrapped in parafilm and put in ziploc with wet towel
Group 1 100pM ppMALAT1
Group 1 | Group 2 | Group 3 | ||||||||
Image | Well | Mark & Find | Image | Well | Mark & Find | Image | Well | Mark & Find | ||
A | C3 | 1 | E | F8 | 15 | I | C6 | 9 | ||
B | D3 | 2 | F | F5 | 14 | J | C7 | 10 | ||
C | F3 | 13 | G | F6 | 7 | K | E7 | 11 | ||
D | C5 | 4 | H | E6 | 8 | L | F7 | 12 |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
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