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*[[Hosuk:Lab_Notes|LabNote]] ===Human Brain Tissue : fixing test=== *Try Human Brain Tissue fixed with 4% Formaldehyde at 4C for 12hr. *Continued from [[Hosuk:LabNotes/2014-10-14|2014-10-14]] ====Procedure==== *10/15 *Wash with PBS once *Add 8uL BS(PEG)9 + 392uL 1x PBS for 1hr at room temp. *Wash with PBS twice, Add Tris pH8.0 for 30min *Wash with PBS twice, 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 171.5uL *#Buffer 20uL *#25mM dNTP 2uL *#2mM a-dUTP 4uL *#High conc. Phi29 0.5uL *Add RCA mix, incubate at 30C for 18hr ===Decoding PGP1F - Agi26k 20gap, 0gap, 0gap with Suppressor=== *Decoding PGP1F with different Padlock Probes **Dish1 : Agi26k_20gap **Dish2 : Agi26k_0gap **Dish3 : Agi26k_0gap with Suppressor *Matt made 2nd rolonies *All 3 dishes were decoded at one time [[File:Diagram_DecodingSamples_2014-10-15.png|400px]] *Preliminary result will be :[[Hosuk:LabNotes/2014-10-20|2014-10-20]]
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