Matt:LabNotes/2014-3-19: Difference between revisions
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===Secondary RCA=== | |||
#Aspirate but don't wash | |||
#'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. | |||
##2.5ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 497.5ul of '''2xSSC/30% formamide''' | |||
##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) | |||
##Add 200ul 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. | |||
##427.5 ul ddH2O, | |||
##50 ul 10x Phi29 buffer, | |||
##5 ul 25 mM dNTP, | |||
##10 ul 2 mM aminoallyl dUTP | |||
##7.5 ul Phi29 DNA polymerase (Epicentre, low concentration) | |||
##*500ul Total Volume | |||
#Add 50ul each well and incubate 20hrs at 30C. (Put in at 4:30pm 3/20/14) | |||
#Wash 1X PBS once | |||
#Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr | |||
#Wash 1X PBS twice | |||
#Add 50ul 1M Tris pH 8.0 @RT for 30min | |||
#Wash 1X PBS twice |
Revision as of 23:04, 20 March 2014
Padlock Probe Concentration Test using Fixed Template
- 96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9)
- Alan already marked locations with good cell density
- Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM
- 2 wells for each
Fixing Template in cells
- Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (50ul 100uM MALAT1_Template + 50ul 20X SSC + 400ul H2O)
- Add 50ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 50ul BS(PEG)9 mix (10ul BS(PEG)9 + 490ul 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 (Started at 4pm)
- Wrapped in parafilm and put in ziploc with wet towel
Negative Control 0pM (C8 & C9)
Component | Volume |
10X Ampligase Buffer | 10 |
5U/ul Ampligase | 10 |
H2O | 80 |
Total | 100 |
10pM (D8 & D9)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
50pM (E8 & E9)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 5 |
5U/ul Ampligase | 10 |
H2O | 75 |
Total | 100 |
100pM (C10 & D10)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
10nM (E10 & F9)
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.
- 2.5ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 497.5ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 200ul 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.
- 427.5 ul ddH2O,
- 50 ul 10x Phi29 buffer,
- 5 ul 25 mM dNTP,
- 10 ul 2 mM aminoallyl dUTP
- 7.5 ul Phi29 DNA polymerase (Epicentre, low concentration)
- 500ul Total Volume
- Add 50ul each well and incubate 20hrs at 30C. (Put in at 4:30pm 3/20/14)
- Wash 1X PBS once
- Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr
- Wash 1X PBS twice
- Add 50ul 1M Tris pH 8.0 @RT for 30min
- Wash 1X PBS twice