Hosuk:LabNotes/2014-5-7: Difference between revisions
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*#*5M Betaine 40uL | *#*5M Betaine 40uL | ||
*#*CircLigaseII 2uL | *#*CircLigaseII 2uL | ||
*#RCA | *#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 | |||
*#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 168uL | |||
*#*Buffer 20uL | |||
*#*25mM dNTP 4uL | |||
*#*2mM a-dUTP 4uL | |||
*#*Phi29 4uL | |||
*#Add RCA mix, incubate at 30C for 20hr |
Revision as of 22:44, 7 May 2014
Test : +/- a-dUTP, and long RT incubation at 4C
- Continued from 05/06
Procedure
- 05/06
- 10% Formalin for 15min at RT(25C) --> done at CERC
- Wash with PBS 3 times --> done at CERC
- Move samples to the lab at PFBH
- Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min at room temp.
- mean while prepare RT mix and put the mix on an ice
- Aspirate 0.25% Triton X-100 in DEPC-PBS and wash with PBS 3 times
- Add RT mix and put in 4C, incubate for 15min. (1hr. for other 2 sample of target specific primers)
- Move samples to 37C and incubate for 12hr.
- 05/07
- Wash with PBS once
- Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL)
- add 200uL BS(PEG)9 mix, incubate for 1hr at RT
- Wash with PBS once, add Tris pH 8.0, incubate for 30min
- 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 3hr.
- 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
- 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 168uL
- Buffer 20uL
- 25mM dNTP 4uL
- 2mM a-dUTP 4uL
- Phi29 4uL
- Add RCA mix, incubate at 30C for 20hr