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A high efficiency method for the preparation of 1,4-piperazinediethanesulfonic acid buffer
Piperazine diethanesulfonic acid PIPES has a variety of buffer systems, among which the buffer solution Transformation Buffer (also called TB solution) can be used to prepare highly efficient competent cells.
Competent state refers to a special physiological state in which cells (or bacteria) can take up DNA molecules from the surrounding environment and are not easily broken down by restriction endonucleases in the cell. Some bacteria are naturally competent, such as Bacillus subtilis, and some are artificially competent, such as Escherichia coli. Treating the recipient cells with a special method can change the permeability of the cell membrane, making it a competent cell that allows carrier molecules with foreign DNA to pass through.
Competent TB solution raw material-PIPES powder
Transformation Buffer preparation:
|Prepare piperazine diethanesulfonic 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 with 5N KOH* low PH insoluble
|Add 10.9g of MnCl2·4H2O with a final concentration of 55mM to make 1 liter
|Filter sterilization, store at 4 degrees
The transformation of competent bacterial cells uses the calcium chloride method. E. coli taken out in liquid nitrogen or low-temperature refrigerator, streak on LB plate, 37 degrees overnight until a single colony grows. Pick a colony with a diameter of 1-3mm to inoculate the SOB medium in a 250ml/3L Erlenmeyer flask or 80ml/1L Erlenmeyer flask. Incubate at 18 degrees at 150-250RPM for 19-50h. Stop the culture when the OD600 is about 0.4-0.8, cool in 4 degrees ice water for 10 minutes, centrifuge at 300 RPM for 15 minutes, recover the bacteria after removing the supernatant, suspend in 1/3 volume of ice-cold TB solution, and centrifuge again for 10 minutes to recover the bacteria. Suspend with 1/12.5 volume of TB, add DMSO with a final concentration of 7%, and then cool for 10 minutes in 0.1-1ml aliquots, and store directly in liquid nitrogen or -80°C.
In the TB solution of suspended Escherichia coli, the main components include buffer PIPES and calcium chloride to transform cells into competent. It should be noted that this method is suitable for E. coli strains that do not contain plasmids, and is not suitable for large plasmids. The transformation efficiency may be reduced after cryopreservation. In addition, the optimum OD600 of different bacterial strains is also different, and needs to be adjusted according to different experiments.
There are many methods for preparing competent cells with TB, and the efficiency of different methods may be different, and the applicable strains or cell types may also be different. The PIPES produced by Desheng is a powder reagent, and competent TB solutions can be prepared according to different needs.
Desheng Biochemical is a manufacturer specializing in the production of biological buffers in China. The buffer products include Tris(Hydroxymethyl)Aminomethane, 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid, 3-Cyclohexylaminopropanesulfonic Acid, etc., all with a purity of over 99%, if needed Business friends of you can contact us!
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