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Preparing Solutions in the Lab

Calculating Molar, Percentage and "X" Solutions

MOLAR SOLUTION: This type of solution is a solution where one 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/mol x 5 moles/liter x 0.1 liter = 29.29 g in 100 ml of solution PERCENT SOLUTIONS: Percentage (w/v) = weight (g) in 100 ml of solution; Percentage (v/v) = volume (ml) in 100 ml of solution. Example: make a 0.7% solution of agarose in TBE buffer, weight 0.7 of agarose and bring up volume to 100 ml with TBE buffer. X SOLUTIONS: Most enzyme buffers are prepared as concentrated solutions- five times or ten times the concentration of the working solution- these are then diluted such that the final concentration of the buffer in the reaction is one times. Example: To set up a restriction digestion in 25 l, one would add 2.5 l of a 10X buffer, the other reaction components, and water to a final volume of 25l.

Preparing Working Solutions from Concentrated Stock Solutions

Most buffers used in molecular biology require the same components but in varying concentrations. Thus to avoid making every single buffer from scratch it is best to make several concentrated stock solutions and dilute them when 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. Use the following equation for calculating amounts of stock solution needed: C i x V i = C f x V f , where C i = initial concentration, or conc of stock solution; V i = initial vol, or amount of stock solution needed C f = final concentration, or conc of desired solution; V f = final vol, or volume of desired solution.

Solution Preparation

Always refer to a lab reference manual for specific instructions on preparation of a particular solution and the bottle label for any specific precaution when handling the chemical. Begin by weighing out the desired amount of chemical. If the amount required is less than o.1g then use an analytical balance. Afterwards place chemical into appropriate size beaker with a stir bar, then add less than the required amount of water. All solutions should be prepared with double distilled water. Observe when the chemical is dissolved and then transfer to a graduated cylinder and add the required amount of distilled water to achieve the final volume. When preparing solutions containing arar or agarose you need to weigh the agar or agarose directly into the final vessel. If a specific pH needs to be used in the solution, check the pH meter with fresh buffer solutions and follow instructions for using a pH meter. If possible autoclave at 121 degrees Celcius for 20 minutes. Remember some solutions can not be autoclaved like SDS therefore these should be filter sterilized through a 0.22 m or 0.45 m filter. For bacterial cultures media must be autoclaved the same day it is prepared , preferably within an hour or two. Afterwards store at room temperature and check if it is contaminated prior to use by holding the bottle at eye level and gently swirling it. Bacterial plates that require solid media can be prepared in advance, autoclaved and stored in a bottle. The agar can be melted in the microwave when needed with any additional components added.

Concentrated solutions

Concentrated solutions such as 1M Tris-HCl pH=8.0, 5M NaCl, can be used to make working stocks by adding double-distilled autoclaved water in a sterile vessel to the appropriate amount of the concentrated solution.

Glassware

Glassware used for molecular biology must be thoroughly clean. If glassware is dirty, contains bacterial contamination or traces of detergent can inhibit reactions or degrade nucleic acid. Rinse glassware with distilled water and autoclave at 150 degrees Celsius for one hour. Experiments with RNA, glassware and solutions are treated with diethyl-pyrocarbonate to inhibit RNases which can be resistant to autoclaving. Polypropylene tubes are turbid and are resistant to many chemicals, like phenol and chloroform. Polycarbonate or polystyrene tubes are clear and not resistant to many chemicals. Make sure that the tubes you are using are resistant to the chemicals used in your experiment. Micro pipet tips and microfuge tubes should be autoclaved before use.

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