IGEM:Hong Kong UST 2014/2009/Notebook/ATP Concentration Effect to Ligation Speed/2014/06/05
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Thursday, 5th of June 2014<html><img src="http://openwetware.org/images/2/26/Team_copy.jpg" width="20%" align="right"></html>
Make 10 mM ATP stock for 1 ml by doing serial dilution. The ATP solution then sterilized with filter sterilization
Make the recipe for ligation. Each person in the team will use 4 micro centrifuge tube to create the control, 1mM ATP, 2mM ATP, and 4 mM ATP. The amount of insert and backbone added varies due to the different amount of concentration of nucleic acid in each set. The ratio of insert to backbone must be 3:1. And since the length of the insert and backbone is 1 kb to 2 kb respectively, the amount of insert must be 30 ng and backbone must be 20 ng. ( total amount of both insert and backbone is ~50 ng)
Incubate in room temperature for 50 minutes
Transform the plasmid into the competent cell DH5α (high efficiency) with 1hr recovery period.
Grow transformed cells on the Chloramphenicol LB plates for 16 hours in 37°C using spread plate method.
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