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Preparation of HEPES buffer with different concentrations
Biological buffer is a solution that can keep the pH of the solution relatively stable when a small amount of acid or alkali is added. Most cells can only move in a narrow pH range, and a buffer system is needed to resist the occurrence of metabolic processes. pH changes. In the process of cell culture, HEPES buffer is often indispensable. In open culture conditions, if HEPES is added, it can prevent the pH of the medium from oxidizing to increase, thereby maintaining the pH at about 7.0. HEPES molecular weight is 238.31, chemical name: 4-hydroxyethylpiperazineethanesulfonic acid, molecular formula: C8H18N2O4S. HEPES is commonly used in biological buffers, pH buffer range: 6.8-8.2, used in biochemical diagnostic kits, DNA / RNA extraction kits and PCR diagnostic kits. Let's take a look at how different concentrations of HEPES buffer are prepared.
Depending on the application, one is pure HEPES + NaOH, and the other is HEPES + salt. The various preparation methods are summarized as follows:
1. Preparation of 1.0 M / L HEPES, pH = 7.0 Stock solution (500ml): Weigh 119.15g HEPES dissolved in 400ml distilled water, add 0.5 ~ 1M / L NaOH aqueous solution to adjust to the required pH It is 6.8 ~ 8.2), then make up to 500ml with distilled water and store at 4 degrees Celsius.
2. HEPES Buffer formula with a small amount of salt (500ml): Weigh out HEPES 6.5g, NaCl 8.0g, Na2HPO4.7H2O 0.198g, adjust to the appropriate pH value with 0.5M / L NaOH aqueous solution, and finally set the volume to 500ml , The HEPES concentration was 55mM / L.
3. Preparation of 2 × HEPES buffered salt solution (100ml): Weigh 1.6g NaCl, 0.074g KCl, 0.027g Na2HPO4.2H2O, 0.2g glucan or dextran and 1g HEPES dissolved in 90ml distilled water, Adjust to the required pH value with 0.5M / L NaOH solution, and then make up to 100ml with distilled water to obtain 42mM / L HEPES.
4. HEPES can be directly added to the prepared culture liquid according to the required concentration, and then sterilized by filtration. Add 2.38 grams of HEPES per 1000ml of culture solution, adjust pH to 7.2 with 1N NaOH after dissolution, filter and sterilize and use. At this time, the use concentration of HEPES is 10 mM / L.
5. It can also be prepared into 100 x storage solution (l mol / L). Before use, 99ml of culture solution is added to 1ml of storage solution, and the final application concentration is still 10mmol / L. l mol / L (100 x) HEPES stock solution preparation method: dissolve 23.8g HEPES in 90ml double-distilled water, adjust pH to 7.5-8.0 with 1N NaOH, then dilute to 100ml with water, filter and sterilize, and separate vials ( 2ml / bottle), store at 4 ℃ or -20 ℃.
The final concentration of HEPES buffer used is 10-50mmol / L, which has no toxic effect on cells. Under normal circumstances, HEPES with a concentration of 20 mmol / L can achieve buffering capacity. The HEPES sold by Desheng Company is about 10g small bottles, and there are also 20g and 50g specifications. You can flexibly prepare according to the actual use.