Hosuk:LabNotes/2015-2-4
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Human Brain Tissue : CA12k_Nov2014 V4 & V7 : 1st Try
- Samples : BA8 on Vectabond treated cover glass mounted at 2014-12-14
- There are only the BA8s on Vectabond treated left.
- 3 BA8 prepared
- S1 : 1st Rolony generation
- S2 : V4 Padlock probe
- S3 : V7 Padlock probe
- CA12k_Nov2014 Padlock Probes
- Made at 2015-01-15 by Matt
- V4 : 1337nM (38ul) --> 7.5uL for 100nM in 100uL Ampligase Mix
- V7 : 803nM (18ul) --> 12.5uL for 100nM in 100uL Ampligase Mix
Procedure
- 02/03
- Take Tissue glass slide from -80C, and put it in a well of sterilized 6 well plate to 50C (hot plate) for 3min. (To prevent RNase activity)
- Add 4% formamide on the glass, incubate for 30min at room temp.
- Wash tissue with 2x SSPE twice for 5min each
- Aspirate 2x SSPE, and wipe solution around the edge of the glass
- Apply adhesive to glass, place glass to MatTek dish, wait for 10~15min for complete dry
- Wash with 2x SSPE twice (add DEPC-PBS on a well, aspirate)
- Add 0.25% Triton X-100 in 2x SSPE for 10 min, Wash with PBS 3 times
- Add - Pepsin incubation 0.01% for 5 min each
- Wash with DEPC-H2O three times
- Add RT mix (Hexamer, -a-dUTP)
- DEPC-H2O 161uL
- M-MuLV Buffer 20uL
- 25mM dNTP 2uL
- 100uM random hexamer 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 overnight.
- 02/04
- Skip BS(PEG)9 step, Wash with PBS once
- Add RNase H mix, incubate for 1hr. at 37C
- 2 ul Riboshredder (Epicentre)
- 10 ul RNase H(Enzymatics)
- 20 ul RNase H buffer(Enzymatics)
- 168 uL H2O
- Wash with nucleaus-free H2O twice
- For 1st Rolony Sample:Add CircLigase II mix, incubate at 60C for 2hr.
- H2O 128uL
- Buffer 20uL
- 50mM MnCl2 10uL
- 5M Betaine 40uL
- CircLigaseII 2uL
- For Ampligase Sample:Add Ampligase mix, incubate at 37C for 30min. then move to 60C for 24hr.
- H2O 72.5uL for V4, 67.5uL for V7
- Buffer 10uL
- Padlock Probe 7.5uL for V4, 12.5 uL for V7
- Ampligase 10uL
- For 1st Rolony generation : 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
- Wash with 2XSSC twice, and 1X SSC twice
- Prepare RCA reaction mix
- H2O 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29(low conc.) 2uL
- Add RCA mix, incubate at 30C overnight
- 02/05
- For V4 & V7 samples: RCA Primer, final concentration : 0.5uM in 200uL (2x SSC) at 60C for 1hr.
- 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
- Wash with 2XSSC twice, and 1X SSC twice
- Prepare RCA reaction mix
- H2O 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29(low conc.) 2uL
- Add RCA mix, incubate at 30C overnight
- For 1st Rolony generation : 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
- 02/06
- For V4 & V7 samples: 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