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

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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)


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