User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/12

Pc-TF Genomics

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08/12/15 Castone Western - gel and transfers Castone Western - gel and transfers Transfections of dCas-Histone + gRNA plasmids were done on 6/25 and 6/27 Protein was prepped using the standard procedure describe at: http://openwetware.org/wiki/Haynes:ProteinExtraction Samples: Luc14: H340_MV11 + g034_pSPgRNA Luc14: H340_MV11 (no gRNA) Luc14: H360_MV11 + g034_pSPgRNA Luc14: H360_MV11 (no gRNA) Luc14: H380_MV11 + g034_pSPgRNA Luc14: H380_MV11 (no gRNA) Gal4EED-luc: H340_MV11 + g034_pSPgRNA Gal4EED-luc: H340_MV11 (no gRNA) Gal4EED-luc: H360_MV11 + g034_pSPgRNA Gal4EED-luc: H360_MV11 (no gRNA) Gal4EED-luc: H380_MV11 + g034_pSPgRNA Gal4EED-luc: H380_MV11 (no gRNA) SDS-PAGE General: Protocol: http://openwetware.org/wiki/Haynes:PAGE Gel: Nupage 4-12% Bis-Tris (12 wells) Buffer: NuPAGE® MOPS SDS Chamber: Invitrogen XCell SureLock Protein standard: PageRuler Plus Pre-stained Protein sample prep: Final volume = 20 μL Sample buffer: 4x Nupage LDS sample buffer 4x sample master mix (x25) 25x 5.0 μL 4x LDS = 125 25x 1.0 μL 1M DTT = 25 Protein samples (12): 14 μL protein + 6 μL master mix Dummy samples (10): 14 μL water + 6 μL master mix Heat @ 100°C/ 5 min.Cool at room temp. Loading - Gel 1: PageRuler Plus Luc14: H340_MV11 + g034_pSPgRNA Luc14: H340_MV11 (no gRNA) Luc14: H360_MV11 + g034_pSPgRNA Luc14: H360_MV11 (no gRNA) Luc14: H380_MV11 + g034_pSPgRNA Luc14: H380_MV11 (no gRNA) dummy dummy dummy dummy dummy Loading - Gel 2: PageRuler Plus Gal4EED-luc: H340_MV11 + g034_pSPgRNA Gal4EED-luc: H340_MV11 (no gRNA) Gal4EED-luc: H360_MV11 + g034_pSPgRNA Gal4EED-luc: H360_MV11 (no gRNA) Gal4EED-luc: H380_MV11 + g034_pSPgRNA Gal4EED-luc: H380_MV11 (no gRNA) dummy dummy dummy dummy dummy Run gels: 120 V, 50 min. Note: next time run 60 min. TRANSFER Electroblot Load both at same time, "feet" facing inward Transblot Turbo transfer pack, mini format, 0.2 um PVDF Bio-Rad Transblot Turbo: Bio-Rad Protocol, Mixed MW, 2.5A, 25V, 7 min (slot A, upper) Note: Wow! worked super well and only took 7 min! However, did not see 10 kD band on the membrane, but 15 kD is there (histones) Poncaeu stain Rinsed blot in di-water and wahed in 1x Poncaeu for ~15 min. Rinsed off excess with di-water Note: Pxi4 imager not set up correctly, troubleshoot tomorrow with Solo Pyon 08/13/15 - RESULTS Blot 1 - Luc samples 1-6 Blot 2 - Gal4EED-luc samples 7-12