BL21(DE3) Competent Cells - FAQs

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.

We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.

Preparation procedures and formulations for all of our competent cells are proprietary. All chemically competent cells are delivered in an aqueous solution that contains a mixture of salts, along with a freezing stabilizer such as glycerol or DMSO.

The recommended heat shock time does increase slightly with increasing volume of competent cells. For a 50 µl reaction volume, you should heat shock at 42°C for 30 seconds. For 100 µl, 45 seconds is recommended and for 250 µl, 60 seconds. It is important to do a positive control transformation of pUC19 along with transformation of your ligation product to accurately determine your relative efficiency of transformation.

It may be surprising, but in most cases transformation efficiency per µg of DNA will actually decrease when higher amounts of plasmid are transformed in one reaction. While you may see more colonies on your plates, much of the extra plasmid DNA you added will actually be wasted. Competent cells eventually become oversaturated with DNA, and adding more plasmid beyond that level will not result in any additional colonies. For example, when transforming 10 pg plasmid DNA, the efficiency of TOP10 cells is 1.0x10E9 colonies per µg of DNA that you added. If you transform 1 ng all at once, the overall efficiency is likely to decrease to ~1.0x 10E8 colonies per µg, and transforming 1 µg in a single reaction will likely result in efficiency less than 1.0 x 10E6 colonies per µg.

To maximize colony yield, it is better to transform smaller amounts of DNA in multiple reactions rather than adding all of the DNA to one reaction. This is most important when transforming a library, where you ideally want each plasmid to be represented by a colony after transformation.

SOC (Super Optimal Catabolite) Medium Preparation (for 1 Liter):

1) To a 2 Liter flask with stir bar add the following:
- Bacto Tryptone 20 g
- Yeast Extract 5 g
- Sodium Chloride (NaCl) 0.58 g
- Potassium Chloride (KCl) 0.186 g
2) Add sterile water to a final volume of 1 Liter.
3) Mix well on magnetic stir plate for 5-10 minutes or until all of the ingredients are well mixed and completely dissolved.
4) Autoclave 30 minutes.
5) Allow to cool to room temperature.
6) Add 10 ml of sterile 2M Magnesium Solution (1M Magnesium sulfate, 1M Magnesium chloride)and mix well.
7) Add 10 ml of sterile 2M Glucose and mix well. (Final Glucose concentration is 20 mM).