Making high electrocompetent coli (DH5a or Top10) - (Dec/29/2004 )

Hi,

I'm looking for an efficient protocol for making high quality electocompetent bacteria in order to get the best results in transformation in the range of 1e10 colonies/µg plasmid or more.

I've found a protocol for TG1 strain on internet but Can I apply the same protocol for DH5a or Top10 ?
Is it possible to get more that 1e10 colonies/µg DNA?

If someone can give me a protocol link or protocol tips for making such electrocmpetent bacteria...

Regards.
David.

Here is the protocol I've found. , it seems that we can get something like 1e10 colonies/µg DNA...

Preparation of competent cells

1. Grow an overnight culture of TG1 (from a fresh minimal plate incubated at 37°C) in 10 ml 2xTYmedium at 30°C.

2. In the morning, dilute the overnight culture: 0.8 ml in 200 ml 2xTY medium (in a 1-liter shake flask) and incubate at 30°C (in a shaker at 265 rpm) until an OD600 of 0.40-0.45.

3. Transfer the cells to 4x 50 ml Falcon tubes and incubate on ice (for 2 h, the longer the better).

4. Centrifuge the cells at 2900 rpm for 8 min in a pre-chilled (SH3000) rotor/centrifuge and discard the supernatant.

5. Wash 1: Resuspend each pellet in 10 ml of cold sterile 1 mM Hepes + 10% glycerol, by pipetting up and down. When the pellet is resuspended, fill the tube to 50 ml. Leave on ice for 10-15 min.

6. Centrifuge the cells at 2900 rpm for 8 min in a prechilled rotor/centrifuge and discard the supernatant.

7. Wash 2: Repeat steps 5 and 6, but don't use the pipette to resuspend the cells. Simply add 50 ml of solution and resuspend by inverting and flicking the tubes.

8. Wash 3: Resuspend each pellet in 5 ml of cold sterile 1 mM Hepes + 10% glycerol (by flicking). Combine the cell solutions by groups of 4. Leave on ice for 10-15 min.

9. Centrifuge the cells at 2900 rpm for 6 min in a prechilled rotor/centrifuge and discard the supernatant.

10. Resuspend the pellet in 450 µl of cold sterile 1 mM Hepes + 10% glycerol (by flicking).

11. The electro-competent TG1 are now ready for use.

12. Testing the efficiency: 100 µl of cells + 30 ng pUC19 should result in > 5x109-2x1010 transformants per µg pUC19.

Electroporation in TG1

1. Add 1 µl of DNA (equivalent of one ligation reaction) per Eppendorf tube.

2. Chill on ice.

3. Add 200 µl freshly prepared electro-competent TG1.

4. Mix by pipetting up and down once. 5. Transfer to a pre-chilled 0.2 µm electroporation cuvette.

6. Incubate on ice for 1 min.

7. Electroporate: Ec3 (3 kV) for bacteria in a Bio-Rad Micropulser (the time-constant should be around 5.8).

8. Immediately add 1.8 ml of SOC medium.

9. Plate out immediately (2 electroporations/large square plate) on 2xTY + 5% glucose + 100 µg/ml carbenicillin-agar plates.

10. Also, take a small sample for titration, to determine the total size of the library.

Ji David
so far as i know, 10 to the 10th transformants per microgram dna is pretty damn good! About the strains - i am not familiar with either TG1 or Top10 and i doubt anyone in this forum is - i suggest you post your question in the molecular biology forum.