Preparation of Solutions

Preparation of Solutions

Calculation of Molar, %, and "X" Solutions

(i) A molar solution is one in which 1 liter of solution contains the number of grams equal to its molecular weight.

Example. To make up 100 mL of a 5M NaCl solution = 58.456 (mw of NaCl) g × 5 moles × 0.1 liter = 29.29 g in 100 mL sol mole liter.

(ii) Percent solutions

Percentage (w/v) = weight (g) in 100 mL of solution

Percentage (v/v) = volume (mL) in 100 mL of solution.

Example. To make a 0.7% solution of agarose in TBE buffer, weigh 0.7 of agarose and bring up the volume to 100 mL with the TBE buffer.

(iii) "X" solutions.

Many enzyme buffers are prepared as concentrated solutions, e.g., 5 X or 10 X (5 or 10 times the concentration of the working solution), and are then diluted so that the final concentration of the buffer in the reaction is 1 X.

Example. To set up a restriction digestion in 25 mL, one would add 2.5 mL of a 10 X buffer, the other reaction components, and water for a final volume of 25 mL.

Preparation of Working Solutions from Concentrated Stock Solutions.

Many buffers in molecular biology require the same components, but often in varying concentrations. To avoid having to make every buffer from scratch, it is useful to prepare several concentrated stock solutions and dilute as needed.

Example. To make 100 mL of TE buffer (10 mM Tris, 1 mM EDTA), combine 1 mL of a 1 M Tris solution and 0.2 mL of 0.5 M EDTA and 98.8 mL sterile water. The following is useful for calculating amounts of stock solution needed:

Ci × Vi = Cf × Vf,

where Ci = initial concentration, or concentration of stock solution

Vi = initial volume, or amount of stock solution needed

Cf = final concentration, or concentration of desired solution

Vf = final volume, or volume of desired solution.

Steps in Solution Preparation:

1. Refer to the laboratory manual for any specific instructions on preparation of the particular solution and the bottle label for any specific precautions in handling the chemical.
2. Weigh out the desired amount of chemical(s). Use an analytical balance if the amount is less than 0.1 g.
3. Pour the chemical(s) in an appropriate size beaker with a stir bar.
4. Add less than the required amount of water. Prepare all solutions with double-distilled water (in a carboy).
5. When the chemical is dissolved, transfer to a graduated cylinder and add the required amount of distilled water to achieve the final volume. An exception is when preparing solutions containing agar or agarose. Weigh the agar or agarose directly in the final vessel.
6. If the solution needs to be at a specific pH, check the pH meter with fresh buffer solutions and follow the instructions for using a pH meter.
7. Autoclave, if possible, at 121°C for 20 minutes. Some solutions cannot be autoclaved; for example, SDS. These should be filter-sterilized through a 0.22-mm filter. Media for bacterial cultures must be autoclaved the same day it is prepared, preferably within an hour or 2. Store at room temperature and check for contamination prior to use by holding the bottle at eye level and gently swirling it.
8. Solid media for bacterial plates can be prepared in advance, autoclaved, and stored in a bottle. When needed, the agar can be melted in a microwave, any additional components, e.g., antibiotics, can be added, and the plates can then be poured.
9. Concentrated solutions, e.g., 1M Tris-HCl pH = 8.0, 5M NaCl, can be used to make working stocks by adding autoclaved double-distilled water in a sterile vessel to the appropriate amount of the concentrated solution.

Glassware:

Glass and plasticware used for molecular biology must be scrupulously clean. Glassware should be rinsed with distilled water and autoclaved or baked at 150°C for 1 hour. For experiments with RNA, glassware and solutions are treated with diethylpyrocarbonate to inhibit RNases, which can be resistant for autoclaving. Plasticware, such as pipettes and culture tubes, is often supplied sterile. Tubes made of polypropylene are turbid and resistant to many chemicals, like phenol and chloroform; polycarbonate or polystyrene tubes are clear and not resistant to many chemicals. Micropipette tips and microfuge tubes should be autoclaved before use.