Transcript Chemistry and Physics of Electrophoresis - Bio-Rad

Chemistry and Physics of Electrophoresis
Bio-Rad Biotechnology Explorer™ Dye/STEM Kit
Instructors - Bio-Rad Curriculum and Training
Specialists
Sherri Andrews, Ph.D.
[email protected]
Damon Tighe,
[email protected]
Leigh Brown, M.A.
[email protected]
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Electrophoresis separates molecules by
CHARGE and SIZE
Electrophoresis means “to carry with electricity”
Electrode
Buffer
with
charged
ions
Molecular
sieve
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Electrode
Electricity
Components of electrophoresis – The Buffer
 Buffer with charged ions
– Must buffer the DNA and not change pH significantly with
increase in temperature
– Must be capable of carrying charge
– Must be capable of solubilizing a gel matrix molecule
– Must maintain an orderly distribution of the electric field
– Must not interfere with future reactions (ie isolation of DNA
fragments, ligation of DNA fragments, cloning of DNA) or worded
differently, must not chemically react with the samples
– Must not heat up too much during a run
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Components of electrophoresis – The Buffer
 Most developments for slab gel electrophoresis
occurred near 1971 and since then…not much has
changed with respect to buffers, electrodes and
molecular sieves…
– Most design was based on Protein gel work and it was
assumed that properties would carry over to DNA
electrophoresis
– Buffers for DNA agarose gel electrophoresis have almost
exclusively been
• TAE – Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol) acetate
EDTA
• TBE – Tris borate EDTA
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Components of electrophoresis – The Buffer –
Tris based
“For reasons not fully
evident today, Tris
became established as
the favored cation for
DNA Electrophoresis.”
J.R. Brody, S.E.
Kern/Analytical
Biochemistry 333 (2004)
1-13
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Components of Electrophoresis – The Buffers
 TAE Buffer
– Pros : Does not interfere with subsequent enzymatic
reactions such as ligations
– Cons: Lower buffering capacity and higher conductivity than
TBE buffer, temperature dependent pH
 TBE Buffer
– Pros: Higher buffering capacity and lower conductivity than
TAE buffer
– Cons: Borate can interfere with downstream DNA enzymatic
reactions due to interactions with sugar groups in DNA,
temperature dependent pH
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Components of Electrophoresis – The Electrodes
 Electrodes must be capable of carrying charge with
minimal chemical transformations occurring while
immersed in a salty solution
– Must be relatively chemically inert in a salty solution
– Must be capable of carrying a charge with a voltage
difference of between 50-300 V DC
– Must be maleable enough to mold to desired dimensions
– Reusable for fairly permanent fixation into an instrument (ie
do not want to have to replace regularly)
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Components of Electrophoresis – The Electrodes
 Commercial electrodes are exclusively made of
platinum
– Pros: High conductivity, Extremely low reactivity
– Cons: Expensive
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Components of Electrophoresis – Molecular
Sieve
 The Molecular Sieve must be capable of separating
molecules via size
– Should be easily moldable
– Should not chemically interact with the molecules being
separated
– Should have a high enough melting point that electrophoretic
runs will not melt it
– If polymeric, should be of molecular purity such that there is
no batch to batch differences
– Must be able to form a variety of pore sizes
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Components of Electrophoresis – Molecular
Sieve
 Most commonly used for horizontal electrophoresis is
agarose
– Complex polysaccharide agar-bearing marine algae
– Neutrally charged, less chemical complexity than agar
(which also contains agaropectin which has heavily modified
acidic side-groups)
– Low likelihood to react with DNA
– Forms pore sizes amenable to separating DNA of 100
basepairs and up in size
– Gelling temperature : 35-40ºC
– Melting temperature : 86-90ºC
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So how do we design an electrophoresis
chamber?
Dye Electrophoresis
Commercial versus built box comparisons
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The Chemistry of Electrophoresis
 Electrolysis always occurs during electrophoresis.
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Electrochemistry in Action
 What other chemical reactions will occur?
– If the buffer is Tris acetate EDTA
• Electrodes made of copper
• Electrodes made of galvanized steel
• Electrodes made of aluminum
– If the buffer is Tris borate EDTA
• Electrodes made of copper
• Electrodes made of galvanized steel
• Electrodes made of aluminum
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Polymer Chemistry in Action
 What is the impact of different percentages of
agarose gels on separation?
 What is the impact of a different polymer on
separation?
– Gelatin versus agarose
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Physics of Electrophoresis
 Simple DC Circuit model
R = Electrophoresis
system
V = Battery tower
V = IR
 Measure Voltage and Current, calculate Resistance
 Determine impact of different buffers on resistance
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Physics of Electrophoresis
 More complex model of resistance – contribution of
electrophoresis components on resistance
Direction of current
buffer
gel
Side view of gel box
R4
V = Battery tower
RTOT
R1
R2
R3
RTOT =
1
(1/(R1+R2+R3) + 1/R4)
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Physics of Electrophoresis – Measurements
versus Calculations
 Use a conductivity meter to measure the conductivity of an
electrophoresis buffer and then convert to conductance
– Conductance = Conductivity (V/m) / kc (conductivity constant)
– For the Vernier probe kc = 1 m-1
 Create a simple DC circuit using the gel box, buffer, leads and
electrodes, and measure the voltage and current of the system
using a multimeter. Calculate the resistance of the
electrophoresis buffer and then convert to conductance.
–
–
Resistance (ohm) = Voltage (V)/current (Amp)
Conductance = 1/Resistance
 Are the measured and calculated values the same?
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Physics of Electrophoresis – Ohmic heating



Energy dissipated per unit time = Energy dissipated per charge passing
through resistor x Charge passing through resistor per unit time
Q I2R which comes from P=VI=I2R=V2/R
Also know that Q = vCT where C is the volume specific heat capacity
of the substance, v is the volume in ml, and T is the change in
temperature
–
–
–
–
–
–
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Joule – a unit of energy
Coulomb – a unit of electrical charge
Volt – a unit of electrical potential (joule per coulomb)
Watt – a unit of power or energy per unit time (joule per second)
Ampere – a unit of current flow (coulomb per second)
Specific heat – the amount of energy required to raise the temperature of a
given volume of a substance 1ºC
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Physics of Electrophoresis – Ohmic heating –
Experimental
 Experimental setup
 Do you have a bomb calorimeter lab? Use it to measure the
specific heat capacity of your buffer and agarose setup!
– Measure the volume of buffer and estimate total volume of
buffer+gel
– Prepare an electrophoresis chamber with your buffer of choice
– Measure the voltage of your power supply (batteries)
– Measure the initial temperature of your system
– Measure the initial current of your system
– Run electrophoresis for 20 minutes
– Measure the final voltage, final temperature, and final current
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Physics of Electrophoresis – Ohmic heating –
Calculations

Pave = Vave x Iave
–

E = Pave x t
–


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Calculate the energy absorbed by the electrophoresis system where v is the volume of the
electrophoresis resistant components (buffer + gel), c is the specific heat capacity of the buffer + gel
(estimated at 4 J/K*ml, slightly less than water…) and T is the difference between the initial and final
temperate in degrees Kelvin (same as the difference in degrees Celsius)
According to the law of conservation of energy, the energy dissipated by the resistor should
equal the energy absorbed by the electrophoresis system. Do your findings support this?
Compute the percent variation between the two values.
Q =vcT
–

Calculate the heat energy dissipated by the electrophoresis system in this process where t is 20
minutes converted into units of seconds
Q =vcT
–

Calculate the average voltage and average current and use these values to calculate the average
power
Or, assuming that all the energy generated by the system was dissipated as heat, calculate the
specific heat capacity of the electrophoresis resistant components for differing buffering systems
Do you find your results match “common knowledge” that TBE heats up less than TAE (has
a higher heat capacity?)
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Challenge!
 From what you know about the chemistry and physics of the
system
– Design a new buffering system which
• Does not react with DNA
• Has a high heat capacity
• Has a high buffering capability
– Design a new electrode system which
• Does not react electrochemically
• Does not cost a lot
– Design a new molecular sieve system which
• Separates molecules of the appropriate size
• Separates molecules in a shorter time
– Put it all together!!
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