Preparation of Solutions Laboratory Methods

a) Calculation of Molar, %, and "X" Solutionsi) A molar solution is one in which 1 liter of solution contains the number

of grams equal to its molecular weight.

ii) Percent solutions w/v) = weight (g) in 100 mL of solutionv/v) = volume (mL) in 100 mL of solution.iii) "X" solutions. Many enzyme buffers are prepared as concentrated solutions,mL, one would addmL of a 10 X buffer, the other reaction components, and water formL.b) Preparation of Working Solutions from Concentrated Stock Solutions. Many buffersi × Vi = Cf × Vf,i = initial concentration, or concentration of stock solutioni = initial volume, or amount of stock solution neededf = final concentration, or concentration of desired solutionf = final volume, or volume of desired solution.

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.

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Percentage (

Percentage (

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.

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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

2.5

a final volume of 25

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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:

C

where C

V

C

V

 c) Steps in Solution Preparation i) Refer to the laboratory manual for any specific instructions onii) Weigh out the desired amount of chemical(s). Use an analytical balanceiii) Pour the chemical(s) in an appropriate size beaker with a stir bar.iv) Add less than the required amount of water. Prepare all solutions withv) When the chemical is dissolved, transfer to a graduated cylinder and   vi) If the solution needs to be at a specific pH, check the pH meter withvii) Autoclave, if possible, at 121°C for 20 minutes. Some solutions cannotmm filter. Media for bacterial cultures must be autoclaved theviii) Solid media for bacterial plates can be prepared in advance, autoclaved,ix) Concentrated solutions, e.g., 1M Tris-HCl pH = 8.0, 5M NaCl, can bed) Glassware. Glass and plasticware used for molecular biology must be scrupulously

preparation of the particular solution and the bottle label for any specific

precautions in handling the chemical.

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if the amount is less than 0.1 g.

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double-distilled water (in a carboy).

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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.

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fresh buffer solutions and follow the instructions for using a pH meter.

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be autoclaved; for example, SDS. These should be filter-sterilized through

a 0.22-

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.

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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.

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used to make working stocks by adding autoclaved double-distilled

water in a sterile vessel to the appropriate amount of the concentrated

solution.

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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.

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