AlanFung:LabNotes/Rolony/2014-3-5: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 22: | Line 22: | ||
*1pM | *1pM | ||
*0.1pM | *0.1pM | ||
====Secondary RCA==== | |||
#Aspirate each well but don't wash | |||
#*F7 well was dried up | |||
#'''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=== | ===Anneal Padlock Probes for 24 hours=== |
Revision as of 21:42, 7 March 2014
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)
- Low Concentration (1pM)
- Ultra Low Concentration (0.1pM)
Fixing Template in cells
- 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
- 100pM
- 1pM
- 0.1pM
Secondary RCA
- Aspirate each well but don't wash
- F7 well was dried up
- 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
- 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
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