User:Robwarden/Notebook/Caspase-3 Activatable Probe/2009/09/11

Caspase-3 Activatable Probe

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PCR Validation Agarose Gel Purification 1% Agarose Run at 110V for 30 min Band appeared in expected location ~1kb Ladders: NEB 100bp, NEB 1kb Digestion 5.0 μL 10X Buffer EcoRI 0.5 μL 100X BSA 30.0 μL PCR Product / 1 μg Vector (1X and 5X) 1.0 μL EcoRI 1.0 μL XhoI 12.5 μL dH2O / to 50 μL Run at 37°C for 1 hr. Reaction Cleanup using Qiagen PCR Cleanup Kit Ligation 2.0 μL 10X T4 Ligase Buffer 2.5 μL Vector Backbone 7.5 μL hgLuc Insert 7.0 μL MQ 1.0 μL T4 DNA Ligase Incubated at RT for 10 min. Killcut with SgrAI for 5 min @ 37°C. Transformation Stbl2 Cells Shook at 30°C for 1:30. Plated on LB-Amp. (250 μL/plate on 2 plates) Incubated overnight at 30°C Replating of phgLuc-5x-DNAe 250 μL/plate on 2 plates