Prepare two dilutions of supercoiled DNA at 1 ng/ul and 5 ng/ul.
Place 1 ul of each DNA dilution in microfuge tubes on ice.
Add 50 ul of competent cells (thawed on ice) to each tube.
After 30 min, incubate the tubes at 42oC for 30 seconds.
Remove tubes from water bath and add 1 ml of LB to each tube (containing
the appropriate antibiotic).
Incubate the tubes at 37oC for 1 hour.
After 1 hour, prepare serial dilutions of cells as follow:
For 1 ng DNA,
10-1 dilution -- add 20 ul of cells to 180 ul LB
10-2 dilution -- add 20 ul of 10-1
dilution to 180 ul LB
10-3 dilution -- add 20 ul of 10-2
dilution to 180 ul LB
For 5 ng DNA,
10-1 dilution -- add 20 ul of cells to 180 ul LB
10-2 dilution -- add 20 ul of 10-1
dilution to 180 ul LB
10-3 dilution -- add 20 ul of 10-2
dilution to 180 ul LB
10-4 dilution -- add 20 ul of 10-3
dilution to 180 ul LB
Plate 100 ul of each dilution on LB + Antibiotic plates.
After overnight incubation at 37ºC, count the number of colonies on each plate.
Number of colonies expected
Transformation Efficiency
1ng DNA
5ng DNA
10-1
10-2
10-3
10-1
10-2
10-3
10-4
109/ug
10000
1000
100
50000
5000
500
50
108/ug
1000
100
10
5000
500
50
5
107/ug
100
10
1
500
50
5
0.5
106/ug
10
1
0
50
5
0.5
0
Note: The number of colonies at each dilution and for each DNA amount should be
consistent to each other. That is, if 1 ng DNA, 10-2 dilution plate gives 10
colonies. Then 10-3 dilution at 5 ng DNA dose should give 5 colonies.
