Preparation of high efficiency receptive state by pipes buffer CAS NO.5625-37-6

1. Factory production, more favorable prices, and regular products are available in stock;

2. Desheng is a manufacturer of a series of biological buffer products, which has a wide range of products and can provide you with more comprehensive services;

3. Desheng has been in this industry for 16 years. Our products are independently developed and manufactured. Biological buffer has high purity and guaranteed product quality.

4. Desheng has been engaged in foreign trade since 2010, and we have our own export right.

Piperazine diethylsulfonic acid pipes has a variety of buffer systems, in which the buffer transformation buffer (also known as TB solution) can be used to prepare highly competent cells.

Receptive state refers to 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 intracellular restriction endonuclease. Some bacteria are naturally receptive, such as Bacillus subtilis, and some are artificially receptive, such as Escherichia coli. After special treatment of recipient cells, the permeability of cell membrane can be changed to become competent cells that allow the passage of carrier molecules with exogenous DNA.

Receptive TB solution raw material - pipes powder

Transformation buffer preparation:

Prepare piperazine diethylsulfonic acid pipes 3.0g 10mm, CaCl2 · 2H2O 2.2g 15mm, KCl 18.6g 250mm, add 950ml of water, adjust the pH to 6.7-6.8 * low pH insoluble with 5N Koh, add 10.9g of MnCl2 · 4H2O with a final concentration of 55mm, dissolve it to 1L, filter and sterilize, and store it at 4 degrees.

Calcium chloride was used to transform competent bacterial cells. E. coli taken out of liquid nitrogen or low-temperature refrigerator shall be scribed on LB plate, 37 degrees overnight until a single colony grows. Select colonies with a diameter of 1-3mm and inoculate them into the sob medium of 250ml / 3L conical flask or 80ml / 1L conical flask. Incubate at 18 degrees 150-250 rpm for 19-50 H. When OD600 is about 0.4-0.8, stop the culture, cool it in 4 ℃ ice water for 10min, centrifuge it at 300rpm for 15min, remove the supernatant, suspend it with 1 / 3 volume of ice cold TB solution, centrifuge it again for 10min, suspend it with 1 / 12.5 volume of TB, add DMSO with the final concentration of 7%, cool it for 10min, repack it with 0.1-1ml, and store it directly in liquid nitrogen or - 80 ℃.

In the TB solution of suspended Escherichia coli, the main functional components include buffer pipes and calcium chloride that converts cells into competent state. It should be noted that this method is suitable for E. coli strains without plasmids, but not for large plasmids, and the transformation efficiency may be reduced after cryopreservation. In addition, the optimum OD600 of different bacterial strains is also different, which needs to be adjusted according to different experiments.

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