Preparation of HEPES buffer with different concentrations

Details:

Biological buffer is a solution that can keep the pH value of the solution relatively stable when a small amount of acid or alkali is added. Most cells can only operate in a very narrow pH range, and a buffer system is needed to resist the pH change in the metabolic process. HEPES buffer is often indispensable in the process of cell culture. In open culture conditions, if HEPES is added, the pH in the culture medium can be prevented from increasing, so as to maintain the pH at about 7.0. HEPES has a molecular weight of 238.31, chemical name: 4-hydroxyethyl piperazine ethanesulfonic acid, and molecular formula: c8h18n2o4s. HEPES is commonly used as biological buffer, with pH buffer range of 6.8-8.2. It is used in biochemical diagnostic kit, DNA / RNA extraction kit and PCR diagnostic kit. Let's take a look at how HEPES buffers with different concentrations are prepared.

According to the use, one is pure HEPES + NaOH and the other is HEPES + salt. Various preparation methods are summarized as follows:

1. Preparation of 1.0m/lhepes, pH = 7.0stocksolution (500ml): weigh 119.15g HEPES

Dissolve in 400ml distilled water, add 0.5 ~ 1m / L NaOH aqueous solution to adjust to the required pH (the effective pH range of HEPES is 6.8 ~ 8.2), then fix the volume to 500ml with distilled water and store at 4 ℃.

2. Hepesbuffer formula with a small amount of salt (500ml): weigh hepes6.5g, NaCl 8.0g and na2hpo4.7h2o0.198g respectively, adjust to the appropriate pH value with 0.5m/l NaOH aqueous solution, and finally fix the volume to 500ml. The concentration of HEPES is 55mm / L.

3、2 × Preparation of HEPES buffer salt solution (100ml): dissolve 1.6g NaCl, 0.074gkcl, 0.027g na2hpo4.2h2o, 0.2g glucan and 1g HEPES in 90ml distilled water, adjust to the required pH value with 0.5m/l NaOH solution, and then fix the volume to 100ml with distilled water to obtain 42mm / L HEPES.

4. HEPES can be directly added into the prepared culture medium according to the required concentration, and then filtered and sterilized. Add 2.38g HEPES to every 1000ml of culture medium, adjust the pH to 7.2 with lnnaoh after dissolution, and use after filtration and sterilization. At this time, the concentration of HEPES is 10 mm / L.

5. It can also be prepared into 100x storage solution (lmol / L). Take 99ml culture solution and add LML storage solution before use

The final application concentration is still 10mmol / L. Preparation method of lmol / L (100x) HEPES storage solution: dissolve 23.8g HEPES in

90ml double distilled water, adjust the pH to 7.5-8.0 with lnnaoh, then fix the volume with water to 100ml, filter and sterilize, sub pack small bottles (2ml / bottle), and store at 4 ℃ or - 20 ℃. The final concentration of HEPES buffer is 10-50mmol / L, which has no toxic effect on cells. Generally, HEPES with a concentration of 20mmol / l can reach the buffer capacity. HEPES sold by Desheng company are about 10g vials, as well as 20g and 50g packages, which can be prepared flexibly according to the actual use.