Contents 1 Materials needed 2 Protocol 3 Supposed Results 4 Actual results 4.1 Further results

Transformation Efficiency [DH5α&BL21]

2014/7/27 To test the efficiency of our competent cells!

Materials needed

• K8 plasmids gradient: 50pg/ul, 20pg/ul, 10pg/ul, 5pg/ul, 0.5pg/ul

• Ice box

• EP tubes 12X

• Competent cell: BL21 & DH5α

• C+ agar plates 12X

• No resistance plates 2X

• 42°C waterbath

• LB media

Protocol

1. Thaw competent cells on ice and then pre-chill by placing the tubes on ice.
2. Pipet 50ul of competent cells into each EP tube.
3. Pipet 1ul of DNA into each tube. Flick the tube gently to mix.
4. Incubate on ice for 30 minutes.
5. Heat-shock the cells, 42°C waterbath, 1 minute.
6. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes.
7. Add 200ul of LB media per tube, 37°C, 2 hours. [150rpm, 12:29am]
8. Pipet 20ul from each tube, spread the mixture evenly across the plate.
9. We also did a control for each group. Except for not being transfected by plasmid, other procedures were all the same with those experimental groups.
10. Incubate these agar plates at 37°C overnight [17h, 2014.7.27 15: 30pm~2014.7.7.28 8:30am]
11. Count the number of colonies on a light field or a dark background. Use the following equation to calculate your competent cell efficiency.

    § (colonies on plate) / ng of DNA plated x 1000ng/μg

Supposed Results

Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA, where "cfu" means "colony-forming unit" and is a measurement of cells.

Here are some sample results:

Actual results

[17h, 2014.7.28 8:30am]

No colonies have grown on any of the plates.

Further results

[30h, 2014.7.28 21:30pm]

For the DH5α cells, except 0.5pg/ul, 10pg/ul and the control group (plates with C+ resistance, competent cell with no plasmid transformed), all the other plates have colonies grown out. We can roughly see that, the plate transformed with 50pg/ul plasmids gives us the maximum of colonies. While the plate grown with bacteria transfected by with 10pg/ul plasmids gives the least number of colonies, even less than that of the 5pg/ul group. This strange phenomenon may be explained by the unevenly mixed bacteria liquid of the 10pg/ul group that was used to spread on the agar plates. For those BL21 bacteria, no colonies have grown out on their agar plates, which might be caused by the relatively lower transformation efficiency they possessed. Till now, we can generally conclude that the transformation efficiency of our competent cell is relatively low, at least compared with those commercialized ones. That’s why we always fail to grow out monoclones before.

And this can be solved by: [summarized by Yongkang Long]

1. SOC medium to recover
2. centrifuge before spread
3. spread more volume of bacteria liquid on the agar plate