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Super competent E. coli prepared with PIPES buffe
PIPES Chinese name piperazine-N,N'-bis(2-ethanesulfonic acid), CAS No. 5625-37-6, molecular weight is 302.368, is a commonly used biological buffer, pH buffer range is 6.1-7.5, insoluble In water, soluble in aqueous NaOH solution. It is commonly used in the preparation of E. coli competent experiments to maintain the pH of the solution so that it will not have significant fluctuations.
Other reagents such as HEPES can be used, but PIPES works best.
Unlike buffers containing bis(2-hydroxyethyl)amino groups (such as Bis-tris, Bicine), PIPES cannot form stable complexes with most metal ions and is suitable for buffers in solution systems containing metal ions. According to the existing research results, PIPES can be used to purify tubulin using phosphocellulose chromatography, to purify recombinant GTP-binding proteins ARF1 and ARF2 by gel filtration, and as a buffer to crystallize transketolase from E. coli. In addition, because PIPES can form free radicals, it is not suitable for use in redox systems. When used in cation exchange chromatography, a low concentration of PIPES buffer is more suitable than a high concentration, because PIPES has a relatively large ionic strength and its pKa value is concentration-dependent.
Preparation of PIPES when preparing super competent E. coli:
1. 0.5mol/LPIPES (pH 6.7): Weigh 15.1g PIPE dissolved in 80ml water, adjust the pH to 6.7 with 5M KOH, and adjust the volume to 100ml (only after adjusting the pH to dissolve), in an ultra-clean bench, use sterile filter to filter Store in sterile reagent bottles at -20℃.
2. LB plate, LA plate, 1 50ml Erlenmeyer flask (containing 10ml LB medium), 2 500ml Erlenmeyer flasks (including 100mlLB), 4 round bottom 50ml centrifuge tubes, a lunch box 1.5ml centrifuge tube, yellow tip 1 box, 1 box of white pipette tips, wrapped in newspaper and sterilized separately.
1. Inoculate DH5α on the LB medium with the inoculation loop and draw lines on the plate, and invert the culture for 12-16h.
2. Pick single clones, inoculate in 10ml LB medium, and cultivate 8-16.
3. Take 4ml and 2ml strains respectively, inoculate 100ml LB medium (500ml triangle flask is required), 18℃, 250r, culture for about 18h, until one bottle of bacteria reaches OD600 value between 0.45-0.6, throw away One bottle, measured once every hour.
4. Place the Erlenmeyer flask in ice for 10 min (the following steps can be placed on ice, the shorter the time in air, the better).
5. Separate the bacteria into four 50ml round bottom centrifuge tubes and centrifuge at 4000C for 4min at 4℃ (the centrifuge needs to be pre-cooled in advance).
6. Discard the supernatant at the end of centrifugation, centrifuge again instantly, and use a pipette to aspirate the residual medium.
7. Add 8ml of Inoue transformation buffer to each centrifuge tube, gently shake and invert to mix (approximately 8ml mark on the centrifuge tube, be sure to mix gently, not blow with a gun).
8. Centrifuge at 4000C for 4 min at 4°C. At the end of centrifugation, the supernatant was discarded, then centrifuged instantaneously, and the residual buffer was removed with a pipette.
9. Add 2ml of Inoue buffer to each tube, and gently shake and mix upside down (be sure to mix gently, not pipette). After mixing, the 4 tubes are combined into one tube.
10. Add 0.6 ml DMSO, mix gently, and place on ice for 10 min.
11. Dispense into 1.5ml centrifuge tubes on ice, each tube containing 200ul, 400ul, etc. (The tip for dispensing can be pre-cooled in the refrigerator). Store at -80℃.
The biological buffer PIPES produced by Hubei Xindesheng Material Technology Co., Ltd. has high purity of finished products, ≥99%, stable production process, timely delivery, and the product can be stored at room temperature.