Buffer Preparation

Ten Steps to Accurate and Reliable Reagent Preparation.

Most solutions for use in the molecular biology laboratory require careful preparation and storage. This guide describes, in detail, the essential steps which must be carried out for the accurate and precise preparation of buffered solutions prior to experimentation. If you prepare solutions which do not require buffering, then the quantitative nature of this guide still applies in order to achieve reliable and reproducible experimental results.

1. All glassware to be used for reagent or buffer preparation should be washed in hot water with detergent and subsequently rinsed thoroughly with deionised water and, preferably, dried.

2. Decide on the volume of reagent or buffer required and calculate the required amount of solute(s) and/or volumes of stock solution(s) required to give the precise final concentrations. This can be done using BiochemicalcTM. Ideally, record all weights or volumes used in your laboratory notebook.

3. Either add (i) minimum volume of deionised water followed by solutes/stock solutions, or, (ii) solute(s)/stock solutions followed by minimum volume of deionised water to a clean, appropriately-sized, beaker.

4. Add a clean magnetic flea and dissolve all components in minimum volume deionised water, with stirring on a magnetic stirrer, adding deionised water as required to bring to approximately 80% the final required volume. Ensure complete dissolution before attempting to pH the solution*.

5. Calibrate a pH meter to span the desired pH range of the buffer under preparation.

6. After calibration, rinse the pH probe with deionised water and then place in the buffer solution under preparation.

7. While stirring, and after all components have dissolved*, adjust the buffer to the desired pH using either 5M HCL or 5M NaOH, as required. Occasionally you may alternatively need to use an organic acid such as acetic, citric or succinic acid for pH adjustment.

8. Once the desired pH has been achieved, quantitatively and carefully transfer the solution to a graduated cylinder. Rinse out the beaker with deionised water from a wash bottle and also transfer this rinsing to the graduated cylinder. Be careful not to add too much water during rinsing or else you may go over the final volume of buffer required in the graduated cylinder.

9. Make up the volume in the graduated cylinder to 100 % with deionised water ensuring the bottom of the meniscus lines up with the 100% mark (e.g., 100 ml or 500 ml etc.). Place clingfilm or parafilm tightly over the top and mix by inversion only (three –four rotations).

10. Transfer the reagent to a clean storage bottle. Label precisely with (i) composition, (ii) your initials, (iii) date of preparation and (iv) storage temperature. If you know the expiry date, then place this information on the label also. State if reagent must be sterile. Store at the required temperature. Example:

 100 mM Maleic acid

150 mM NaCl

pH 7.5

Your Initials 19/12/11

Store at 4 oC


* occasionally, complete solute dissolution requires pH adjustment (e.g., casein).