User:Robwarden/Notebook/Caspase-3 Activatable Probe/2009/09/08

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PCR Validation Agarose Gel Purification 1% Agarose Run at 110V for 30 min Band appeared in expected location ~500bp 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. Gel Purification Ran on 1% agarose at 110V for 25 min. Strong bands seen & removed for each digestion. Purified with qiagen kit. Concentration determined by running 1μL against a ladder. No DNA visible for insert -> undetectable concentration 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 Cell Lines: 1X -> Top10 5X -> Stbl2 Shook at 30°C for 1:30. Plated on LB-Amp. Incubated overnight at 37°C (1X) and 30°C (5X)