Stock Preparation in C3

General

1. Before handling each chemical check for specific hazards/features, e.g. light sensitivity, corrosive nature of chemical, volatility, etc.
2. Use the appropriate care when handling every chemical and use gloves/facemask if needed.
3. All stock solutions are made using water as the solvent, unless otherwise stated. All water used to make up stock solutions is to be ultrapure (18mΩ.cm: ‘MilliQ’ or equivalent).

Solids

Molar Solutions

Using the molecular weight of the compound, calculate the amount of solid to be weighed out for a 50 mL quantity at the desired concentration – two decimal places are generally sufficient. The solid is dissolved in water, the pH adjusted as appropriate, and made up to a 50 mL volume using a measuring cylinder. The solution is sterile filtered (0.22 µm syringe filter) into a clean, labelled 50 mL centrifuge tube.

Example: Make up 50 mL of a 3.5 M ammonium sulfate solution (ammonium sulfate MW= 132.14 g mol-1)

For 50 mL volume need

(3.5 mol l-1 x 132.14 g mol-1)/1000 mL l-1 x 50 mL = 23.13g

solid to be weighed out.

This is dissolved in approximately 40 mL of water with gentle heating and stirring and water added to a final volume of 50 mL. The solution is sterilised by passing through a 0.22 µm syringe filter.

If the solid has waters of hydration then the molecular weight of hydrated solid must be used. For example, magnesium sulfate can be purchased as an anhydrous salt (MgSO4, CAS 7487-88-9) with a molecular weight of 120.366 g mol-1. Magnesium sulfate is more commonly found as the heptahydrate
(MgSO4·7H2O, CAS 10034-99-8) with a molecular weight of 246.47 g mol-1. To make up 50 mL of a 1 M
solution one would use 6.02 g of the anhydrous form OR 12.32 g of the heptahydrate form.

%w/v Solutions

These are made up in reference to the weight of compound per 100 mL of final volume. This is the standard method of making up solutions where the compound has an ambiguous molecular weight –
polyethylene glycols, for example, or salts with mixed hydration states (usually denoted ·xH2O)

Example: Make up 10 %w/v NaCl

Dissolve 10 g of NaCl in approximately 90 mL of water then add water to a final volume of 100 mL (measuring cylinder accuracy). The solution is sterilised by passing through a 0.22 µm syringe filter.

Liquids

%v/v Solutions

These solutions are made up in reference to the volume of liquid within the final 100 mL of solution.

Example: 35 %v/v ethanol

Water is added to 35mL ethanol until the total volume is 100 mL. For a 50 ml volume of this stock
solution, 17.5 mL of ethanol would be made up to the 50 ml volume with water.

Example: 80 %v/v PEG 400

An 80 mL volume of the neat PEG liquid is decanted into a measuring cylinder – to reduce the
viscosity of the PEG, heat gently in a microwave or by placing the bottle in warm water. The volume is made up to 100 mL with water. The cylinder is sealed well with Parafilm and then carefully mixed by multiple inversions. This solution is sterilized using the Steriflip® (Millipore) tube-top filter units with Duropore PVPF 0.22 µm membrane.

%w/v Solutions

These solutions are made up in reference to the weight of liquid within the final 100 mL of solution. Notice that for a liquid with a density of 1 g mL-1 %v/v and %w/v are equivalent.

Example: 10 %w/v glycerol

This stock is made by weighing 10 g of glycerol into a measuring cylinder and adding water to a 100mL volume.

Example: 50 %w/v PEG 600

This solution is made up by gently warming PEG 600 until it becomes liquid (water bath) and weighing out 50 g into a 100 mL measuring cylinder and adding water to 95 mL. Mix by inversion, then bring up to a final volume of 100 mL with water. Thorough mixing is required prior to filtering through a Steriflip® (Millipore) tube-top filter units with Duropore PVPF 0.22 µm membrane.

Calibrate the pH meter with the two standard buffers that span the desired pH, e.g., sodium malonate pH 5 buffer is measured using the pH probe after calibration with standard pH buffers 4 and 7.

Use the conjugate acid or base solution at the appropriate concentration to adjust the pH for the solution if it is available.

Example: 50 mL of 1 M sodium acetate pH 4.5

The solution is made by dissolving up 4.10g of sodium acetate (MW 82.03 g mol-1) in approximately 40 mL water. Water is added to a final volume of 50 mL. Then adjust the pH down to 4.5 with 1 M acetic acid.

Example: A solution of 3.4M sodium malonate pH 7.0

The solution is made by mixing a 3.4 M sodium malonate solution with a 3.4 M malonic acid solution until the pH 7 is reached.

If there is no obvious conjugate partner then use concentrated hydrochloride acid to adjust the pH down and a freshly prepared concentrated sodium hydroxide solution (NaOH pellets dissolved up in a small amount of water) for alkaline pH adjustments.

Example: Tris buffers

These are made by dissolving TRIZMA base in water and adjusting the pH with concentrated HCl to the desired pH, before adjusting the final volume.

Example: HEPES buffers

These are made by dissolving HEPES in water and adjusting the pH with concentrated fresh NaOH to the desired pH, before adjusting the final volume.

Some acid/base pairs to be used in buffer preparations are listed below:

• Sodium malonate / malonic acid
• Sodium, ammonium or potassium phosphates / phosphoric acid (or use the monobasic/dibasic pair – e.g. NaH2PO4/Na2HPO4 )
• Sodium, ammonium or potassium acetates / acetic acid
• Sodium, ammonium or potassium citrates / citric acid
• Trizma / conc. HCl
• HEPES / NaOH
• MES / NaOH
• Sodium cacodylate / HCl

pH Nomenclature

Given the accuracy of pH measurements in general, we do not record pH measurements beyond one decimal place.

Polyethylene glycol (PEG) Stocks

Due to the deterioration of the PEG stocks caused by light and heat, these stocks are stored at -20°C in the freezer.

Only those PEG stocks which are used frequently and in larger quantities are stored unfrozen, and then only 50 mL is thawed at any one time. The unfrozen stocks are stored at 4°C in the dark in the coldroom. Analysis of our PEG usage showed that the commonly used PEG stocks are PEG 3350, PEG 4000, PEG 6000 and PEG 8000. If the 4°C stock is used up, defrost another tube, and write the date of thawing onto the label. Stocks which have been at 4°C for longer than 6 months are discarded.

When you need to use a frozen PEG stock, defrost the relevant tube and mark on the lid a stroke so that you can tell both which tube to select next time but also to record how many freeze/thaw cycles the stock has been through. After use, return the PEG stock to the freezer.

Should there not be enough volume in the first tube, defrost a second tube, mark its lid and decant the extra solution required into the first tube. The 2nd tube should then be returned to the freezer