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

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 * style="background-color: #EEE"|[[Image:Penn_fulllogo.gif|128px]] Caspase-3 Activatable Probe
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Agarose Gel Purification

 * 1% Agarose
 * Run at 110V for 30 min
 * Band appeared in expected location ~1kb
 * Ladders: ,

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


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