Agarose Gel Electrophoresis

What does gel electrophoresis do? employs electromotive force to move molecules through a porous gel separates molecules from each other on the basis of

size and/or charge and/or shape

basis of separation depends on how the sample and gel are prepared

Why perform electrophoresis on ds DNA?

To separate fragments from each other To determine the sizes of fragments To determine the presence or amount of DNA To analyze restriction digestion products

Ethidium bromide stained gel photographed on UV light box in black and white

Length standards

power supply

gel box

Pouring a horizontal agarose gel

Where does the current come from? A direct current power supply Ions supplied by the buffer The charge on the macromolecules being

separated Electrolysis of water

Where does the current come from? Electrolysis of water

4H2O 2H2 + O2 + 2H2O

self-ionization of water throughout the buffer: 4H20 4H+ + 4OH-

At the negative pole• 4H+ + 4e- 2H2

At the positive pole• 4OH- O2 + 2H2O + 4e-

At which electrode would you expect more bubbles? Why?

Basics of Gel Electric Circuits

V (volts) = I (milliamps) X R (resistance) For a segment of a gel/buffer system

cross-sectional area of buffer or gel, resistance strength of buffer = [ion], resistance most resistance is in the agarose gel itself

What factors affect mobility of linear ds DNA?

Pore size of the gel [agarose] pore size pore size friction mobility

Voltage across the gel voltage mobility

Length of the DNA molecule smaller molecules generate less friction and so move

faster Ethidium bromide (stain) intercalated into DNA

decreases charge to mass ratio and so decreases mobility

Why don’t charge and shape affect mobility of linear ds DNA? all DNA molecules have an essentially identical

charge to mass ratio 1 negative charge/phosphate and 1phosphate/base,

so . . . . charge is directly proportional to length

different lengths have essentially identical rod shape

Note: shape does affect mobility of circular and/or single strand DNA or RNA

Visualization Monitoring the progress of the electrophoresis

tracking dyes visible to naked eye during run xylene cyanol (migrates with ~5.0 kb fragments) bromphenol blue (migrates with fragments of a few

hundred base pairs) Orange G (migrates with fragments of ~50 bp)

but mobility of tracking dyes can vary substantially depending on agarose concentration type

Visualization Locating the DNA fragments in the gel

ethidium bromide staining mutagen, wear gloves! visible under UV light wear UV opaque face or eye shield to observe!

Locating the DNA fragments in the gel comparison to length standards

Ethidium bromide stained gel photographed on UV light box in black

and white

Length standards

Factors affecting resolution

Resolution = separation of fragments The “higher” the resolution, the more space between

fragments of similar, but different, lengths Resolution is affected by

agarose type agarose concentration salt concentration of buffer or sample amount of DNA loaded in the sample voltage

What is agarose? linear carbohydrate polymer extracted from

seaweed , agarbiose

forms a porous matrix as it gels shifts from random coil in solution to structure in

which chains are bundled into double helices

What is agarose? (cont’d) multiple types of agarose

Standard agarose - LE Gels at 35-38oC; Melts at 90-95oC Becomes opaque at high concentrations Makes a fairly sturdy gel

Low melting agarose (NuSieve) Gels at 35oC; Melts at 65oC

• Often used to isolate DNA fragments from gel Modified by hydroxyethylation to lower M.P. Is relatively translucent at high concentrations Makes a fragile gel

Intermediate forms or combinations of LE and NuSieve can provide sturdy, translucent gels at high agarose concentrations Good for resolving smaller fragments

Resolution of ds linear DNA fragments in agarose gels

% Agarose (w/v) Size Range (kb prs) for Optimal Separation

0.5 2-300.75 0.7-201.0 0.5-101.5 0.2-32.0 0.1-23.0 (Nu-Sieve) 0.07-1.54.0 (N-S) 0.04-0.95.0 (N-S) 0.03-0.66.0 (N-S) 0.01-0.4

0.7% 2.5%

Effect of agarose concentration on linear DNA fragment resolution.The two lanes contain identical DNA samples.

4M 1M 0M

Effect of salt concentration on resolution of fragments.Samples in all three lanes are identical except for [salt].

0.1 0.25 0.5 1.0 5.0 DNA Hind III fragments (g)

Effect of sample DNA concentration on resolution.

Voltage voltage, rate of migration to increase the voltage

increase the setting on the power supply increase the resistance

decrease the gel thickness decrease the ion concentration

if voltage is too high, gel melts as voltage is increased, large molecules migrate at a rate

proportionally faster than small molecules, so lower voltages are better for resolving large fragments but the larger ds DNA fragments are always slower than the smaller ones

Buffer Systems Remember, buffer systems include weak acids and/or bases

that do not dissociate completely. If ions resulting from dissociation are “removed,” more weak

acid and/or base will dissociate. Purposes of buffer

Keep solution at pH compatible with molecules being separated Generate ions consistently to

maintain current keep resistance low

Both gel and the solution in the gel box are buffered.

Buffer Systems (cont’d)

Two commonly used buffers for routine agarose gel electrophoresis TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA

Tris (T) is a weak base. Acetic (A) acid and boric (B) acid are weak acids.

Acetic acid is more completely ionized at pH 8.0 than is boric acid, so TBE has a high buffer capacity than TAE.

Buffer Systems (cont’d) TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA

loses buffer capacity during long or high voltage gel runs; • anode end of gel becomes acidic• gel may melt from the increased resistance that results from ion depletion

resolves high MW fragments better than TBE

TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA higher buffer capacity somewhat more expensive resolves low MW fragments better than TAE may interfere with subsequent reactions

Non-denaturing agarose gel loading solutions

Composition tracking dyes

are used to follow progress of electrophoresis sometimes interfere with later visualization of DNA

a solute to increase density so that sample falls to bottom of loading well with minimal dilution solute examples: glycerol, Ficoll

Other gel types, with different purposes, use different loading solutions!

Ethidium bromide staining

Binds to DNA by intercalation between stacked bases lies perpendicular to helical axis makes Van der Waals contacts with bases above and below

+ charge, so migrates toward negative pole reduces the charge to mass ratio of the DNA fragment to which

it is bound Because of the change in charge to mass ratio, it alters DNA

mobility, especially of circular covalently closed DNA

Ethidium bromide staining Used to visualize DNA with UV light

uv 254 nm absorbed by DNA and transmitted to EtBr excitation at 302 or 366 nm fluorescence at 590 nm

560 nm = visible red/orange >/= 10ng/band required for visualization

Bound dye fluoresces 20-25X more than dye in solution because of fixed position of planar group proximity of dye to bases

UV light damages eyes and skin! Wear goggles and/or face shield.

Trouble shooting Smearing

torn sample wells voltage too high for large fragments too much DNA

Use </= 0.5 ug / fragment / 0.25cm2 migration area Gel melts

voltage too high ionic strength too low

Poor resolution wrong [agarose] small bands are fuzzy – the gel run may have been too long at too low

a voltage, allowing diffusion of the DNA and broadening of the band