Centrifugation, a separating technique

Centrifugation

Centrifugation is a basic separation technique. A centrifuge is a device for separating particles in an applied centrifugal field in a solution.

There are two different forces act on an object moving in a circular motion.

Centrifugal force:

Force directed outward from the center. E. g. While turning a bus in twist way, the passengers strike on the bus wall is due to centrifugal force.

Centripetal force:

The force exerted towards the center is now as centripetal force. E. g. the force act on passengers by the turning car

Now, suppose a particle is exerted to sediment by centrifugal force, then

• The rate or velocity at which it sediments is proportional to the force applied


• Sedimentation is more rapid when the force applied is greater than the gravitational force of the Earth


• Basis of separation is to exert a larger force than does the Earth’s gravitational force.

Basic Principle of Sedimentation

The particles to be separated are suspended in a specific liquid media, held in tubes or bottles which are located in rotor in centrifuge machine, positioned centrally to the drive shaft. These particles are differing in size, shape and density.

As we have already mentioned that,

The rate of sedimentation is dependent upon the applied centrifugal field (G)

G = W²R

Where:

W-Angular velocity of revolving particle (Remember: one revolution of the rotoe is equal to 2 radians)

R- Radial distance from axis of rotation

In terms of revolution per minute, we have W= 2p rev min^-1/ 60

Therefore:

G = W²R

= 4p² (rev min^-1)² R / 3600

It is expressed as a multiple of the earth’s gravitational field (g=981 cm s^-2)

Hence

RCF, Relative Centrifugal Field=G / g

= 4p² (rev min^-1)² R / 3600 x 981

= 1.119 x 10^-5(rev min^-1)² R

= x g unit (number times g)

It means, RCF is the ratio of the weight of the particle in the applied centrifugal field to the weight of the same particle when acted by gravity alone. Therefore the rotor speed, radial dimensions and time of the rotor must be quoted during the centrifugation.

However:

This is not the only case in Biochemical experiments as biological samples are always found in dissolved or suspended form in a solution. Thus, the rate of sedimentation not only depends on the centrifugal field but also on

1. Mass of particle

2. Density of particle

3. Density and viscosity of the medium used

4. The extent to which its shape deviates from spherical

Now according to Newton’s Second law of Motion, the centrifugal force exerted on particle is

F = M. a

= M. W²R

Where:

M: mass of particle

a: acceleration while in angular motion= W²R

Increasing the sharpness of a turn, w r decreases. Since r is linear w has greater effect on the particle.

It causes the molecules to sediment down the centrifuge tube. They start to move downward to sediment; however they encounter opposing force, a frictional resistance in their movement.

Frictional force = f. dr/dt

= 6phR_p (dr/dt)

Where:

f- Frictional force

dr/dt- rate of sedimentation expressed as the change in radius with time (velocity v)

h-Viscosity coefficient of medium

R_p- radius of sedimenting particle

The sedimenting molecule must also displace the solvent into which it sediments and give rise to a buoyant force

Buoyant force= mass x a

= V. d_m W²R

Where:

V- Specific volume of the molecule

d_m – Density of the medium

While sedimenting, the velocity of the particle increases until equals the frictional force resisting its motion through the medium. This is an equilibrium state when the particles stop to move or sediment.

Centrifugal force = Frictional force + Buoyant force

M. W²R = 6phR_p (dr/dt) + V. d_m W²R

4/3p R_p³ d_p W²R = 6phR_p (dr/dt) + 4/3p R_p³ d_m W²R

4/3p R_p³ (d_p- d_m) W²R = 6phR_p (dr/dt)

dr/dt = 2/9h R_p² (d_p- d_m) W²R

v = 2/9h R_p² (d_p- d_m) W²R

dr/dt = v, is the velocity of the sedimenting particle

Mass = Deansity x Volume

d_p = Density of particle

d_m = Density of medium

From above equation, it seems clear that velocity is proportional to its size, to the differences in density between the particle and medium and to the applied centrifugal field. It is zero when the density of the particle and medium are equal. It decreases when the viscosity of the medium increases.

Since the R_p is in square form, the size of particle has greater influence on velocity.

For a particle, h, R_p^, d_p^, d_m and W all are constants

t = 9h/ [2W²R_p² (d_p- d_m) ] In R_b/R_t

Where

t = the sedimentation time in seconds

R_t = radial distance from the axis of rotation to liquid meniscus

R_b = radial distance from the axis of rotation to bottom of tube

It is now clear that a mixture of heterogeneous approximately spherical particles can be separated by centrifugation on the basis of their densities, their sizes and etc.

t µ 1 / R_p²

It means, higher the size particles, faster is the sedimentation of it and smaller the size slower is the sedimentation (takes longer time).

Centrifuges and their uses

1. Low Speed Centrifuge

· Least expensive and simplest in many design

· Maximum rotor speed of 4000-6000rpm (3000-7000 X g)

a) Small bench centrifuges

· To collect small amounts of materials (250mm³⁾ that is rapidly sediment (1-2 min)

· No special cooling system

· Ambient air flows around the rotor to cool the system

· Use to rapid sedimentation of blood samples

b) Large capacity refrigerated centrifuges

· Refrigerated rotor chambers for cooling the sample

· Large volumes 10, 50 and 100 cm³ processing depending upon the rotors and tubes

· Maximum capacity of 1.25 dm³

· Rotors are mounted on a rigid suspension

· Erythrocytes, coarse or bulky precipitates, yeast cells, nuclei and chloroplasts

2. Microcentrifuge

· Maximum rotor speed of 12000rpm with RCF of 10000g

· Have total capacity of 1.5ml over very short time (0.05-5 min)

· Use to sediment large particles like cell ppt

3. High speed refrigerated centrifuge

· Maximum rotor speed of 25000rpm with RCF of 60000g

· Have total capacity of 1.25 dm³

· Interchangeable fixed angle and swinging buckets rotors

· Use to collect microorganisms, cellular debris, larger cellular organelles and proteins precipitates by ammonium sulphate

· Not use for viruses and smaller organelles like ribosome

4. Continuous flow centrifuge

· Relatively simple and high speed centrifuge

· Special design rotor (long and tubular) with non interchangeable system

· Have total capacity of 1-1.25 dm³/min with continuous flow

· Particles sediment at wall and excess clarified medium overflows through an outlet port

· Use to collect bacterial and yeast cells from their mass culture of about 100-500 dm³

5. Ultracentrifuge

· Powerful with speed

· 2 types

a) Preparative ultracentrifuge

• Maximum rotor speed of 30000-80000rpm with RCF of 600000g


• Highly sophisticated with refrigerated, sealed and evacuated to minimize excess heat generate


• More sophisticated temperature monitoring system employing an infrared temperature sensor


• Overspeed control system to prevent operation of rotor above its max rated speed


• Vibration minimize system (a flexible drive shaft system) during unequal loading of the centrifuge tubes


• Enclosed in heavy armour plating


• Airfuse for some biochemical applications requiring high centrifugal force


• Use for sediment macromolecule/ligand binding kinetic studies, steroid hormone receptor assays, separation of major lipoprotein from plasma and deproteinisation of physiological fluids for amino acid analysis

b) Analytical ultracentrifuge

• Maximum rotor speed of 70000rpm with RCF of 500000g


• Highly protective chambers with refrigerated and evacuated system also have an optical system to enable the sedimenting material to be observed throughout the process.


• Three types of optical system, a light absorption system, alternative Schlieren system and Rayleigh interferometric system (both measures refractive index of solution)

Design and types of Preparative Rotors

• These are rotating instruments in Centrifuges


• During rotation at high speed, higher stress forces generated


• Made up of aluminum alloy and titanium alloy which don’t rust-brass, steel or Perspex


• Can tolerate nearly twice the centrifugal force of rotors


• Protective coating to the metal surface by anodizing or by applying black epoxy paint


• Various types of rotors

a) Swing bucket rotors

· Staring off in a vertical position but during acceleration of the rotor swing out to horizontal position

CENTRIFUGATION . RCF CALCULATION

The relative centrifugal force (RCF) can be calculated from the following equation:

RCF = (1.119 x 10-5) (rpm)²(r)

Where rpm is the speed of rotation expressed in revolutions per minute and r (radius) is the distance from the axis expressed in cm. The RCF units are "x g" where g represents the force of gravity. RCF can also be determined from the NOMOGRAPH below. Place a straight edge to intersect the radius and the desired RCF to calculate the needed rpm. Alternatively place the straight edge on the radius and the rpm to calculate the g-force. For example, spinning a sample at 2500 rpm in a rotor with a 7.7 cm radius results in a RCF of 550 x g.

SEPARATION METHODS IN PREPARATIVE ULTRACENTRIFUGATION

1. Differential Centrifugation: The process of differential centrifugation is based on the fact that organelles have differences in size, shape and density. As a result, the effect of gravity on each is different. We can use this principle to separate an organelle from a homogenous solution of particles by artificially controlling the gravity of a solution. This is done by putting the solution in a variable speed centrifuge and rotating them at a high rate of speed. This creates a force that can be much greater than the force of gravity, and particles that would normally stay in solution will fall out and form a pellet at the bottom of the tube.

Differential centrifugation schemes involve stepwise increases in the speed of centrifugation. At each step, more dense particles are separated from less dense particles, and the successive speed of centrifugation is increased until the target particle is pelleted out. The final supernatant is removed, the pellet is resuspended and further study or purification can be done on it. The fractionation of rat liver is an example of how this process works:

2. Density Gradient Centrifugation

Density gradient centrifugation is a technique that allows the separation of cells, organelles and macromolecules, depending on their size, shape and density.

A density gradient is created in a centrifuge tube by layering solutions of varying densities with the dense end at the bottom of the tube. Cells and large molecules are usually separated on a shallow gradient of sucrose or other inert carbohydrates even at relatively low centrifugation speeds, while macromolecules such as proteins and nucleic acids are separated at higher centrifugation using ultracentrifuges.

Criteria for an ideal density gradient centrifugation medium are:

• the additive must form a solution within the required density range


• the additive must not interfere with, or damage, the sample


• the solvent must be compatible with the sample


• the solution must have a refractive index within the practical range, as well as a low viscosity


• The additive must be easily removable from the sample.

The additives for density gradient centrifugation can be divided into four main categories:

1. Salts of Alkali Metals

These solutions fulfill most of the above requirements. However, due to the high ionic strength, hydrogen bonding within biological macromolecules (protein, nucleic acid - protein complexes) is impaired by a chaotropic effect. Therefore these salts are mainly used for DNA and RNA separations. Cesium chloride is used most frequently. Other useful salts include sodium iodide, sodium bromide, cesium sulfate and cesium acetate. Potassium tartrate has been used to separate viruses from host cells.

It should be kept in mind that the density of the sample is highly dependent on the hydration of the macromolecule, which in turn depends to a large extent on the dehydration power of the salt solution.

2. Neutral, Water-Soluble Molecules

In this class of compounds sucrose is most widely used. It has a useful density range of up to 1.29. This range can be increased to 1.37 by addition of glucose or by dissolving sucrose in D₂O. Sucrose has very little effect on macromolecules, but affects enzyme activity. Due to its high osmotic pressure, sucrose solution dehydrates cells and their organellae very efficiently. Glycerol solutions are the preferred media for the separation of enzymes because they do not affect enzyme activity. They exhibit a high viscosity, requiring prolonged centrifugation times. More importantly however, glycerol penetrates biological membranes.

3. Hydrophilic Macromolecules

Dextran gradients have been used for the separation of microsomes. Separations achieved with dextrans show similar results to those obtained by using synthetic sucrose/ epichlorohydrin co-polymers. In some cases bovine serum albumin has been applied, but the preparation of an appropriate solution is very difficult.

4. Synthetic Molecules

These additives are the sodium or methyl glucamine salt of triiodobenzoic acid and of metrizoic acid. It should be kept in mind that the parent acid of these salts may precipitate on adjusting the pH to acidic values. Metrizamide, a covalently bonded compound of glycosamine and metrizoic acid is most widely used. This additive forms solutions of relatively low viscosity. These solutions are stable over a wide range of pH and ionic strength, and show practically no interference with the analytes.

Density gradient centrifugation methods are of two types, the rate zonal technique and the isopycnic (isodensity or equal density) technique.

Rate Zonal Technique: When mixtures of cellular extracts are layered on top of a density gradient in a tube and subjected to centrifugation, the various components move through the gradient at different rates that are dependant on their sizes and shapes. These different components appear as distinct bands or zones in the gradient with large components migrating farthest in the tube in a given period of time. The rate with which a fraction moves the fixed distance in the gradient tube is dependant of its sedimentation value (S) that, in turn is determined by the size and shape of that fraction. By comparing the different position of the components in the gradient, it is possible to make an approximate measurement of their molecular weight. It is, however, difficult to precisely determine these molecular weights, as this requires knowledge about the shape of these molecules, which is hard to determine with accuracy. This density gradient separation technique is called rate zonal centrifugation and is usually performed with a shallow sucrose gradient. The different components being separated by this technique are denser than any of the sucrose concentrations used in the gradient. Samples are, therefore, centrifuged just long enough to separate the components of interest. Longer centrifugation than necessary would allow all components to form a pellet at the bottom of the tube. One of the most important applications of this technique over the past decades was the separation of transfer RNA (4S) from ribosomal RNA that forms three different classes with distinct sedimentation values 23S, 16S and 5S. This helped to facilitate the characterization of the protein synthesizing system.

Isopycnic Technique: A second density gradient technique, called equilibrium density-gradient centrifugation is used to separate cellular components on the basis of their buoyant density. In this case the cellular mixture is centrifuged through a steep density gradient that contains a high concentration of sucrose, or more often, cesium chloride (CsCl). In these gradients, the molecules being studied have a density somewhere in between the highest and lowest densities of sucrose or CsCl generated in the gradient. The components of a sample begin to move down this gradient in the same way as they do in a rate-zonal density gradient. When a component of the mixture reaches a point where the density of the solution is equal to its own density, it stops moving further and forms a distinct band. The position of the band in the tube is characteristic of the buoyancy of that component. Buoyancy or buoyant density of a substance is its tendency to float in a medium, which in this case is the density gradient. Hence soluble proteins which have similar density (p=1.3 g cm^-3 in sucrose solution) can not usually be separated by this method, whereas subcellular organelles (e.g. Golgi apparatus p=1.11 g cm^-3, mitochondria p=1.19 g cm^-3 and peroxisomes p=1.23 g cm^-3in sucrose solution can be effectively separated.

Equilibrium density gradient centrifugation using CsCl was for decades the method of choice in the purification of highly pure plasmid DNA. Meselson and Stahl, who developed this technique, were the first to use it in an experiment that provided evidence for the semi-conservative replication of DNA and confirmed the double helix structure of DNA proposed by Crick and Watson.

MOST USED SOLUTIONS

Agarose 1%

-weigh 2 g agarose in a 500 ml bottle -ad 200 ml 1* TAE buffer -in the magnetron (H₂) 600 W, 3 min. or autoclave 20 min 120°C -ad 2 µl ethidiumbromide (10 mg/ml) -keep it at 60 °C.

Agarose 1.5%

-weigh 3 g agarose in a 500 ml bottle -ad 200 ml 1* TAE buffer -in the magnetron (H₂) 600 W, 3 min. or autoclave 20 min 120°C -ad 2 µl ethidiumbromide (10 mg/ml) -keep it at 60 °C.

APS 10%

make a 10% (w/v) solution of AmmoniumPeroxodiSulfat (mw 228.2) in milliQ (1 gram in 10 ml) and store 0.5 ml alliquots at -20°C.

CTAB 5%

CTAB (hexadecyltrimethylammoniumbromide) 5 g in 100 ml 120 mM K₂HPO₄ pH 8.0 chloroform + isoamylalcohol 24 : 1.

EDTA 0.5 M (pH 8.0)

Solve 186.1 g Na₂EDTA-2H₂O (mw = 372.24) in 700 ml aqua by adjusting the pH to 8.0 with 10 M NaOH (appr. 45 ml), add bidest to make the volume 1 liter.

Ethidium bromide

10 mg ethidium bromide in 1 ml H₂0

EDTA 0.5 M pH=8.0

Dissolve 186.1 g Na₂EDTA.2H₂O in 700 ml water,

adjust pH to 8 with 10 M NaOH (± 50 ml)
add H2O to make 1 liter
Autoclave 20 min 120°C store at room temperature

Gel-dye TTGE/DGGE

0.05 g bromophenol blue in 10 ml 1* TAE

K2HPO4 buffer solution pH 8.0

make 120 mM K₂HPO₄ (MW = 174.18) 20.9 g/l

make 120 mM KH₂PO₄ (MW = 136.09) 16.3 g/l

ad 947 ml K2HPO4 and 53 ml KH2PO4 together , adjust the pH with

one of the solutions

Loading buffer (6*) DNA TTGE DGGE

0.05% (w/v) bromophenol blue (0.05 g)

40% (w/v) sucrose (40 g)

0.1 M EDTA pH=8 (20 ml 0.5M EDTA)
0.5% (w/v) SDS (0.5 g)
adjust volume to 100 ml

NaAc 3M

40.82 g SodiumAcetat -trihydrat (mw 136.08) in 100 ml aqua

NaOH 10 M

dissolve 400 g Sodium hydroxide (mw 40.0) in 450 ml H2O. Add H2O to 1 liter.

PCI

phenol + chloroform + isoamylalcohol 25 : 24 : 1 (put some buffer on top)

Tris-Cl 1 M (pH 8.0)

solve 121 g Tris base (mw = 121.14) in 800 ml aqua adjust the pH to 8.0 with conc.HCl (appr. 45 ml) , add bidest to make the volume 1 liter

TE (ph 8.0)

10 mM Tris-Cl 10ml/l 1M Tris-Cl pH 8.0 1 mM EDTA 2ml/l 0.5M EDTA pH 8.0

TAE buffer 50*

242.0 g Tris-base

57.1 ml Acetic Acid glacial 100 ml 0.5 M EDTA or 37.2 g Na2EDTA.2H2O Check pH (=8) and adjust volume to 1000 ml with dH2O Autoclave 20 min 120°C store at room temperature.

TAE running buffer 1*

Mix 20 ml 50* TAE with 980 ml H2O

Molarities and Specific Gravities of Concentrated Acids and Bases^a

Acid/base		Molecular weight	% by weight		Molarity (approx.)		Specific gravity	1M solution (ml/liter)
Acetic acid (glacial)		60.05	99.6		17.4		1.05	57.5
Ammonium		35.0	28		14.8		0.90	67.6
hydroxide
Formic acid	46.03			90		23.6	1.205	42.4
				98		25.9	1.22	38.5
Hydrochloric acid	36.46			36		11.6	1.18	85.9
Nitric acid	63.01			70		15.7	1.42	63.7
Perchloric acid	100.46			60		9.2	1.54	108.8
				72		12.2	1.70	82.1
Phosphoric acid	98.00			85		14.7	1.70	67.8
Sulfuric acid	98.07			98		18.3	1.835	54.5
a CAUTION: Handle strong acids and bases carefully.

Acid precipitation solution

1 M HCl

0.1 M sodium pyrophosphate

Nucleic acids can also be precipitated with a 10% (w/v) solution of trichloroacetic acid (TCA); however, this recipe is cheaper, easier to prepare, and just as efficient.

Ammonium hydroxide, concentrated stock solution

See Table A.2A.1.

Ammonium acetate, 10 M

Dissolve 385.4 g ammonium acetate in 150 ml H₂O Add H₂O to 500 ml

Ammonium sulfate, saturated

76 g ammonium sulfate 100 ml H₂O Heat with stirring to just below boiling point Let stand overnight at room temperature

ATP, 100 mM

1 g ATP (adenosine triphosphate) 12 ml H₂O Adjust pH to 7.0 with 4 M NaOH Adjust volume to 16.7 ml with H₂O Store in aliquots indefinitely at -20 C

BBS (BES-buffered solution), 2x

50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES; Calbiochem) 280 mM NaCl

1.5 mM Na₂HPO₄, pH 6.95 800 ml H₂O Adjust pH to 6.95 with room temperature 1 N NaOH H₂O to 1 liter Filter sterilize through a 0.45-um nitrocellulose filter (Nalgene) Store in aliquots at -20 C (can be frozen and thawed repeatedly)

The pH of this solution is critical (pH 6.95 to 6.98). When a new batch of 2x BES buffer is prepared, its pH should be checked against a reference stock prepared (and tested) earlier

BSA (bovine serum albumin), 10% (w/v)

Dissolve 10 g BSA (e.g., Sigma) in 100 ml H₂O. Filter sterilize using a low-protein-binding 0.22-um filter. Store indefinitely at 4 C.

Lower-concentration stock solutions (e.g., 1%), which are useful for various applications, can be made by diluting 10% stock appropriately with sterile water.

BSA is available in various forms that differ in fraction of origin, preparation, purity, pH, and cost; the most commonly used is fraction V. Use the form that is appropriate for the application; this may need to be optimized empirically.

CaCl₂, 1 M

147 g CaCl₂·2H₂O H₂O to 1 liter

Carbonate buffer

1. 1.6 g Na₂CO₃ (15 mM final)

2. 2.9 g NaHCO₃ (35 mM final)

0.2 g NaN₃ (3.1 mM final)
H₂O to 1 liter
Adjust to pH 9.5

CAUTION: Sodium azide is poisonous; follow appropriate precautions for handling, storage, and disposal.

CMF-DPBS (calcium-and magnesium-free Dulbecco's phosphate-buffered saline)

8.00 g NaCl (0.137 M)

1. 0.20 g KCl (2.7 mM)

2. 2.16 g Na₂HPO₄·7H₂O (8.1 mM)

0.20 g KH₂PO₄ (1.1 mM)
H₂O to 1 liter
Store at room temperature

Denhardt solution, 100x

10 g Ficoll 400 10 g polyvinylpyrrolidone 10 g bovine serum albumin (Pentax Fraction V; Miles Laboratories) H₂O to 500 ml Filter sterilize and store at -20 C in 25-ml aliquots

DEPC (diethylpyrocarbonate)-treated solutions

Add 0.2 ml DEPC to 100 ml of the solution to be treated. Shake vigorously to dissolve the DEPC. Autoclave the solution to inactivate the remaining DEPC.

CAUTION: Wear gloves and use a fume hood when using DEPC, as it is a suspected carcinogen.

Many investigators keep the solutions they use for RNA work separate to ensure that "dirty" pipets do not go into them.

Do not treat solutions containing Tris with DEPC, as Tris inactivates the DEPC.

DMEM (Dulbecco's modified Eagle medium), supplemented

Dulbecco's modified Eagle medium, high-glucose formulation (see APPENDIX 2B; e.g., Life Technologies), containing:

5%, 10%, or 20% (v/v) FBS, heat inactivated (optional; see recipe below) 1% (v/v) nonessential amino acids 2 mM L-glutamine 100 U/ml penicillin 100 ug/ml streptomycin sulfate Filter sterilize if anything nonsterile has been added Store up to 1 month at 4 C

DMEM containing this set of additives is sometimes called "complete DMEM." The percentage of serum used is indicated after the medium name--e.g., "DMEM/5% FBS." Absence of a number indicates no serum is used. DMEM is also known as Dulbecco's minimum essential medium.

Ham's F-12 nutrient mixture (APPENDIX 2B; available commercially, e.g., from Life Technologies), is sometimes added to DMEM; the resulting medium is known as DMEM/F-

12.

Because of the higher bicarbonate content, DMEM requires ~10% CO₂ to maintain pH 7.4.

Culture media containing glutamine and penicillin should be warmed to 37 C as few times as possible since components, especially glutamine, degrade rapidly at 37 C.

DPBS (Dulbecco's phosphate-buffered saline)

8.00 g NaCl (0.137 M)

0.20 g KCl (2.7 mM)

0.20 g KH₂PO₄ (1.1 mM)

1. 0.10 g MgCl₂·6H₂O (0.5 mM)

2. 2.16 g Na₂HPO₄·7H₂O (8.1 mM)

0.10 g anhydrous CaCl₂ (0.9 mM) H₂O to 1 liter

DTT (dithiothreitol), 1 M

Dissolve 1.55 g DTT in 10 ml water and filter sterilize.
Store in aliquots at -20 C.

EDTA (ethylenediaminetetraacetic acid), 0.5 M (pH 8.0)

Dissolve 186.1 g disodium EDTA dihydrate in 700 ml water. Adjust pH to 8.0 with 10 M NaOH (~50 ml; add slowly). Add water to 1 liter and filter sterilize.

Begin titrating before the sample is completely dissolved. EDTA, even in the disodium salt form, is difficult to dissolve at this concentration unless the pH is increased to between 7 and

8.

Ethidium bromide, 10 mg/ml

Dissolve 0.2 g ethidium bromide in 20 ml H₂O Mix well and store at 4 C in dark

CAUTION: Ethidium bromide is a mutagen and must be handled carefully.

FBS (fetal bovine serum)

Thaw purchased fetal bovine serum (shipped on dry ice and kept frozen until needed). Store 3 to 4 weeks at 4 C. If FBS is not to be used within this time, aseptically divide into smaller aliquots and refreeze until used. Store 1 year at -20 C. To heat inactivate FBS, heat serum 30 min to 1 hr in a 56 C water bath with periodic gentle swirling during the first 10 to 15 min to ensure uniform heating.

Repeated thawing and refreezing should be avoided, as it may cause denaturation of the serum.

Heat-inactivated FBS (FBS that has been treated with heat to inactivate complement protein and thus prevent an immunological reaction against cultured cells) is useful for a variety of purposes. It can be purchased commercially or made in the lab as described above.

HBSS (Hanks' balanced salt solution)

0.40 g KCl (5.4 mM final)

0.09 g Na₂HPO₄·7H₂O (0.3 mM final)

1. 0.06 g KH₂PO₄ (0.4 mM final)

2. 0.35 g NaHCO₃ (4.2 mM final)

0.14 g CaCl₂ (1.3 mM final)

0.10 g MgCl₂·6H₂O (0.5 mM final)

1. 0.10 g MgSO₄·7H₂O (0.6 mM final)

2. 8.0 g NaCl (137 mM final)

1.0 g D-glucose (5.6 mM final)

0.2 g phenol red (0.02%; optional)
Add H₂O to l liter and adjust pH to 7.4 with 1 M HCl or 1 M NaOH
Filter sterilize and store up to 1 month at 4 C

HBSS may be made or purchased without Ca²⁺ and Mg²⁺ (CMF-HBSS). These components are optional and usually have no effect on an experiment; in a few cases, however, their presence may be detrimental. Consult individual protocols to see if the presence or absence of these components is recommended.

Bottles should be kept tightly closed to prevent CO₂ loss and subsequent alkalinization.

HCl, 1 M

Mix in the following order:

913.8 ml H₂O

86.2 ml concentrated HCl

HeBS (HEPES-buffered saline) solution, 2x

16.4 g NaCl

11.9 g HEPES acid

0.21 g Na₂HPO₄ 800 ml H₂O Titrate to pH 7.05 with 5 M NaOH Add H₂O to 1 liter Filter sterilize through a 0.45-um nitrocellulose filter Store in 50-ml aliquots at -20 C

If the solution is to be used for transfection, the pH should be between 7.05 and 7.12, and should be tested for transfection efficiency.

KCl, 1 M

74.6 g KCl H₂O to 1 liter

LB medium

Per liter:

10 g tryptone 5 g yeast extract 5 g NaCl 1 ml 1 M NaOH Autoclave 25 min

Although the pH is adjusted to near 7 with NaOH, the medium is not very highly buffered, and the pH of a culture growing in the medium drops as the culture nears saturation.

The medium may also contain antibiotics (e.g., 50 ug/ml ampicillin, 12 ug/ml tetracycline), galactosides (e.g., 20 ug/ml Xgal, 0.1 mM IPTG), or other nutritional supplements added after the medium has been autoclaved.

To make LB agar for LB plates, add 15 g/liter agare.

MgCl₂, 1 M

20.3 g MgCl₂·6H₂O H₂O to 100 ml

MgSO₄, 1 M

24.6 g MgSO₄·7H₂O H₂O to 100 ml

MOPS buffer

1. 0.2 M MOPS [3-(N-morpholino)-propanesulfonic acid], pH 7.0

2. 0.5 M sodium acetate

0.01 M EDTA

Store in the dark and discard if it turns yellow

NaCl, 5 M

292 g NaCl H₂O to 1 liter

NaOH, 10 M

Dissolve 400 g NaOH in 450 ml H₂O Add H₂O to 1 liter

PBS (phosphate-buffered saline)

10x stock solution, 1 liter:

80 g NaCl 2 g KCl 11.5 g Na₂HPO₄·7H₂O 2 g KH₂PO₄

Working solution, pH ~7.3: 137 mM NaCl

1. 2.7 mM KCl

2. 4.3 mM Na₂HPO₄·7H₂O

1. 1.4 mM KH₂PO₄

2. 8.00 g NaCl (0.137 M)

1. 0.20 g KCl (2.7 mM)

2. 0.24 g KH₂PO₄ (1.4 mM)

3. 1.44 g Na₂HPO₄ (0.01 M) H₂O to 1 liter

PCR amplification buffer, 10x

500 mM KCl 100 mM Tris·Cl, pH 8.3 (see recipe below) x mM MgCl₂ 0.1% (w/v) gelatin Store in aliquots at -20 C

This solution can be sterilized by autoclaving. Alternatively, it can be made from sterile water and stock solutions, and the sterilization omitted.

15 mM MgCl₂ is the concentration (x) used for most PCR reactions. However, the optimal concentration depends on the sequence and primer of interest and may have to be determined experimentally (see APPENDIX 3).

PMSF (phenylmethylsulfonyl fluoride), 100 mM

Dissolve 0.174 g PMSF in 10 ml of 100% ethanol, isopropanol, or methanol. Store in aliquots up to 2 years at -20 C.

CAUTION: Phenylmethylsulfonyl fluoride is toxic.

Make fresh dilutions from the alcohol stock for each use, because the half-life of PMSF in aqueous solution is <30 min at room temperature and a few hours on ice.

If PMSF is being added to a solution without detergent, the solution should be stirred vigorously during PMSF addition because PMSF has a tendency to form an insoluble precipitate in aqueous solution.

Polylysine-coated tissue culture surfaces

Prepare a stock solution by dissolving 100 mg polylysine in 100 ml water (poly-L-lysine or poly-D-lysine can be used; check specific protocol for choice of isomer) and filter sterilize through a 0.22-um filter. Store in 5-ml aliquots at -20 C. When ready to use, dilute 1 part stock solution with 19 parts water to prepare a 50 ug/ml working solution.

To coat culture dishes, multiwell plates, or chamber slides: Fill tissue culture dishes, multiwell plates, or slide wells with the working solution and incubate 1 hr in a 37 C incubator, then remove solution by vacuum aspiration and allow surface to dry.

To coat coverslips: Sterilize coverslips by autoclaving or by incubating them in 95% ethanol and drying before coating. Place coverslips in a single layer in a petri dish containing working solution and incubate 1 hr at 37 C. Remove coverslips using sterile forceps and allow surface to dry.

Store coated tissue culture ware up to 3 months at 4 C. Use diluted solutions only once.

Potassium acetate buffer, 0.1 M

Solution A: 11.55 ml glacial acetic acid per liter (0.2 M) in water.

Solution B: 19.6 g potassium acetate (KC₂H₃O₂) per liter (0.2 M) in water.

Referring to Table A.2A.2 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 100 ml. Filter sterilize if necessary. Store up to 3 months at room temperature.

This may be made as a 5-or 10-fold concentrate by scaling up the amount of sodium acetate in the same volume. Acetate buffers show concentration-dependent pH changes, so check the pH by diluting an aliquot of concentrate to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.2, prepare closest higher pH, then titrate with solution A.

Potassium phosphate buffer, 0.1 M

Solution A: 27.2 g KH₂PO₄ per liter (0.2 M final) in water.

Solution B: 34.8 g K₂HPO₄ per liter (0.2 M final) in water.

Referring to Table A.2A.3 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 200 ml. Filter sterilize if necessary. Store up to 3 months at room temperature.

This buffer may be made as a 5-or 10-fold concentrate simply by scaling up the amount of potassium phosphate in the same final volume. Phosphate buffers show concentration-dependent changes in pH, so check the pH of the concentrate by diluting an aliquot to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.3, prepare closest higher pH, then titrate with solution A.

Saponin, 10% (w/v)

Dissolve 1 g saponin in 10 ml PBS (see recipe above) Store in 500-ul aliquots at -20 C

Once thawed, the 10% solution is stable for several months when stored at 4 C.

SDS, 20% (w/v)

Dissolve 20 g SDS (sodium dodecyl sulfate or sodium lauryl sulfate) in H₂O to 100 ml total volume with stirring. Filter sterilize using a 0.45-um filter.

It may be necessary to heat the solution slightly to fully dissolve the powder.

SDS electrophoresis buffer, 5x

1. 15.1 g Tris base

2. 72.0 g glycine

5.0 g SDS Distilled, deionized H₂O to 1 liter Store up to 1 month at 0 to 4 C Dilute to 1x before use

Do not adjust the pH of the stock solution; the pH is 8.3 when diluted to 1x. Use purified SDS if appropriate.

SED (standard enzyme diluent)

20 mM Tris·Cl, pH 7.5 500 ug/ml bovine serum albumin (Pentax Fraction V) 10 mM -mercaptoethanol Store at 4 C for up to 1 month

Sodium acetate, 3 M

Dissolve 408 g sodium acetate trihydrate (NaC₂H₃O₂·3H₂O) in 800 ml H₂O Adjust pH to 4.8, 5.0, or 5.2 (as desired) with 3 M acetic acid (see Table A.2A.1) Add H₂O to 1 liter Filter sterilize

Sodium acetate buffer, 0.1 M

Solution A: 11.55 ml glacial acetic acid per liter (0.2 M) in water.

Solution B: 27.2 g sodium acetate (NaC₂H₃O₂·3H₂O) per liter (0.2 M) in water.

Referring to Table A.2A.2 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 100 ml. Filter sterilize if necessary. Store up to 3 months at room temperature.

This may be made as a 5-or 10-fold concentrate by scaling up the amount of sodium acetate in the same volume. Acetate buffers show concentration-dependent pH changes, so check the pH by diluting an aliquot of concentrate to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.2, prepare closest higher pH, then titrate with solution A.

Sodium phosphate buffer, 0.1 M

Solution A: 27.6 g NaH₂PO₄·H₂O per liter (0.2 M final) in water.

Solution B: 53.65 g Na₂HPO₄·7H₂O per liter (0.2 M) in water.

Referring to Table A.2A.3 for desired pH, mix the indicated volumes of solutions A and B, then dilute with water to 200 ml. Filter sterilize if necessary. Store up to 3 months at room temperature.

This buffer may be made as a 5-or 10-fold concentrate by scaling up the amount of sodium phosphate in the same final volume. Phosphate buffers show concentration-dependent changes in pH, so check the pH by diluting an aliquot of the concentrate to the final concentration.

To prepare buffers with pH intermediate between the points listed in Table A.2A.3, prepare closest higher pH, then titrate with solution A.

SSC (sodium chloride/sodium citrate), 20x

3 M NaCl (175 g/liter)

0.3 M Na₃citrate·2H₂O (88 g/liter) Adjust pH to 7.0 with 1 M HCl

STE buffer

10 mM Tris·Cl, pH 7.5 10 mM NaCl 1 mM EDTA, pH 8.0

TAE (Tris/acetate/EDTA) electrophoresis buffer

50x stock solution:

242 g Tris base

57.1 ml glacial acetic acid

37.2 g Na₂EDTA·2H₂O H₂O to 1 liter

Working solution, pH ~8.5:

40 mM Tris·acetate 2 mM Na₂EDTA·2H₂O

TBE (Tris/borate/EDTA) electrophoresis buffer, 10x

108 g Tris base (890 mM)
55 g boric acid (890 mM)
960 ml H₂O
40 ml 0.5 M EDTA, pH 8.0 (20 mM final; see recipe above)

TBS (Tris-buffered saline)

100 mM Tris·Cl, pH 7.5 (see recipe below)
0.9% (w/v) NaCl
Store up to several months at 4 C

TE (Tris/EDTA) buffer

10 mM Tris·Cl, pH 7.4, 7.5, or 8.0 (or other pH; see recipe below) 1 mM EDTA, pH 8.0 (see recipe above)

TEA (triethanolamine) solution

50 mM triethanolamine, pH ~11.5 0.1% (v/v) Triton X-100

0.15 M NaCl

Add Triton X-100 as a 10% stock sterilized by Millipore filtration and stored in the dark to prevent photooxidation (stock is stable 5 years at room temperature). (see recipe below).

TEN (Tris/EDTA/NaCl) solution

40 mM Tris·Cl, pH 7.5 (see recipe below) 1 mM EDTA, pH 8.0 (see recipe above) 150 mM NaCl

TM buffer, 10x

100 mM Tris·Cl, pH 8.0 100 mM MgCl₂

Tris-buffered saline (TBS)

100 mM Tris·Cl, pH 7.5 (APPENDIX 2)
0.9% (w/v) NaCl (150 mM)
Store up to several months at 4 C

Tris·Cl, [tris(hydroxymethyl)aminomethane], 1 M

Dissolve 121 g Tris base in 800 ml H₂O Adjust to desired pH with concentrated HCl Adjust volume to 1 liter with H₂O Filter sterilize if necessary Store up to 6 months at 4 C or room temperature

Approximately 70 ml HCl is needed to achieve a pH 7.4 solution, and ~42 ml for a solution that is pH 8.0.

IMPORTANT NOTE: The pH of Tris buffers changes significantly with temperature, decreasing approximately 0.028 pH units per 1 C. Tris-buffered solutions should be adjusted to the desired pH at the temperature at which they will be used. Because the pK_a of Tris is 8.08, Tris should not be used as a buffer below pH ~7.2 or above pH ~9.0.

Triton X-100, 10% (w/v)

1 g Triton X-100 H₂O to 10 ml Stir to dissolve Filter sterilize through a 0.45-um filter Store protected from light up to 6 months at room temperature

TTBS (Tween 20/TBS)

Dissolve 0.1% (w/v) polyoxyethylenesorbitan monolaurate (Tween 20) in TBS (see recipe above). Store up to several months at 4 C.