Piperazine diethyl sulfonic acid pipes has many kinds of buffer systems, among which transformation buffer (TB solution) can be used to prepare highly efficient competent cells.

Receptive state is a special physiological state in which cells (or bacteria) can absorb DNA molecules from the surrounding environment and are not easy to be decomposed by restriction endonucleases in cells. Some bacteria are naturally susceptible, such as Bacillus subtilis, and some are artificially susceptible, such as Escherichia coli. After special treatment, receptor cells can change the permeability of cell membrane and become competent cells that allow the carrier molecules of exogenous DNA to pass through.

The raw material of TB solution -- pipe powder

Transformation buffer preparation:

Piperazine diethyl sulfonic acid 3.0g 10mm, CaCl2 · 2H2O 2.2g 15mm, KCl 18.6g 250mm were prepared. 950ml water was added, pH was adjusted to 6.7-6.8 * with 5N Koh, insoluble at low pH, and then 10.9g MnCl2 · 4H2O with final concentration of 55mm was added to 1 L, filtered and sterilized, and stored at 4 ℃.

Calcium chloride was used to transform competent bacteria. Liquid nitrogen or E. coli taken out of the refrigerator were crossed on LB plate, 37 ℃ overnight until a single colony grew. The colonies with diameter of 1-3 mm were inoculated into 250 ml / 3 L conical flask or 80 ml / 1 l conical flask of sob medium. Culture at 18 ℃ 150-250 rpm for 19-50 H. When OD600 is about 0.4-0.8, stop the cultivation, cool it in 4 ℃ ice water for 10 min, centrifuge it at 300 rpm for 15 min, remove the supernatant, suspend it with 1 / 3 volume of cold TB solution, centrifuge it again at 10 min, suspend it with 1 / 12.5 volume of TB, add 7% DMSO, cool it again for 10 min, pack it with 0.1-1ml, and store it directly in liquid nitrogen or - 80 ℃.

In the TB solution of E. coli suspension, the main components include buffer pipes and calcium chloride which can transform cells into competent state. It should be noted that this method is suitable for E. coli strains without plasmids, but not for large plasmids. In addition, the optimal OD600 for different bacterial strains is also different, which needs to be adjusted according to different experiments.

There are many ways to prepare receptor cells with TB. The efficiency of different methods may be different, and the suitable strains or cell types may also be different. The pipes produced by Desheng is a powder reagent, which can prepare the sensitive TB solution according to different needs.