Hosuk:LabNotes/2014-7-4
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NCIH-1975 with T48 RT Primer[edit]
- Matt's RT Primers (48 primers) for hybridizing only mRNA (hopefully)
- S1 : T48 RT primer, S2 : rHexamer
Procedure[edit]
- 07/02
- Fix NCIH1975 by adding 10% formalin incubating at RT for 15min, wash with PBS three times
- Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min, wash with PBS three times
- Add 0.1N HCl and incubate 1min, wash with PBS three times
- Wash with 2 ml PBS three times
- Add RT mix (Hexamer, -a-dUTP)
- DEPC-H2O 161uL
- M-MuLV Buffer 20uL
- 25mM dNTP 2uL
- 100uM RT Primer 5uL
- Rnase Inhibitor 2uL
- M-MuLV reverse transcriptase 10 uL
- Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for 12 hr.
- 07/03
- Wash with PBS once, skip BS(PEG)9
- Add RNase H mix, incubate for 1hr. at 37C
- RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
- Wash with nucleaus-free H2O twice
- Add CircLigase II mix, incubate at 60C for 2.5hr.
- H2O 128uL
- Buffer 20uL
- 50mM MnCl2 10uL
- 5M Betaine 40uL
- CircLigaseII 2uL
- RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr. --> 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
- 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
- Aspirate and add 2XSSC, incubate at 60C for 10min
- Aspirate and Wash with 2X SSC, 1X SSC and PBS once each
- Prepare RCA reaction mix
- H2O 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29 2uL
- Add RCA mix, incubate at 30C for 19.5hr
- 07/04
- Wash with PBS once, Prepare 4uL of BS(PEG)9 in 196uL PBS, add 200uL BS(PEG)9 mix, incubate for 1hr at RT
- Wash with PBS once, add Tris pH 8.0, incubate for 30min
- Wash with PBS once
- Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
- Preheat detection Probe at 80C for 5min
- Add to sample and incubate at RT for 10min
- Wash two times for 1 minute each with 1 mL 1X PBS
- Imaging
Result[edit]
- Both samples have most of rolonies inside nuclear, so much localized…
- it cased maybe fixation condition or cell culture condition.
- Random hexamer sample has more rolonies than T48 RT primer has.
- Counts (S1:T48, S2 Random Hexamer)
File:NCIH1975 Count Table S1.jpg File:NCIH1975 Count Table S2.jpg
File:NCIH1975 S1 S2 20x.jpg
File:NCIH1975 S1 S2 63x.jpg
File:NCIH1975 S1 S2 63x Zoom4.jpg