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



11. Making the ATP Stock

Make 10 mM ATP stock for 1 ml by doing serial dilution.
1. Weigh out 0.06 grams of powdered ATP and dilute it using 1 ml of water.
2. Pipette 100 µl of the diluted ATP powder and transfer it into a micro centrifuge tube.
3. Add 900 µl of double distilled water to make the 10mM ATP stock.

The ATP solution then sterilized with filter sterilization

12. Ligation

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)


Control
1mM ATP
2mM ATP
4mM ATP
Insert
30.0 ng
30.0 ng
30.0 ng
30.0 ng
Backbone
20.0 ng
20.0 ng
20.0 ng
20.0 ng
10x T4 buffer (contain 1mM of ATP) (µl)
-
5.0
5.0
5.0
10 mM ATP (µl)
-
-
5.0
15.0
T4 Ligase (µl)
0.5
0.5
0.5
0.5
ddH2O
Adjust to total volume
Adjust to total volume
Adjust to total volume
Adjust to total volume
Total volume (µl)
50.0
50.0
50.0
50.0

Incubate in room temperature for 50 minutes

13. Transformation
Transform the plasmid into the competent cell DH5α (high efficiency) with 1hr recovery period.

14. Culture
Grow transformed cells on the Chloramphenicol LB plates for 16 hours in 37°C using spread plate method.



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