Chemistry and Physics of Electrophoresis

1. Chemistry and Physics of Electrophoresis Bio-Rad Biotechnology Explorer™ Dye/STEM Kit

2. Instructors - Bio-Rad Curriculum and Training Specialists Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Damon Tighe, damon_tighe@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

3. Electrophoresis separates molecules by CHARGE and SIZE Electrophoresis means “to carry with electricity” Electrode Buffer with charged ions Molecular sieve Electricity Electrode

4. 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

5. 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

6. 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

7. 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

8. 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)

9. Components of Electrophoresis – The Electrodes • Commercial electrodes are exclusively made of platinum • Pros: High conductivity, Extremely low reactivity • Cons: Expensive

10. 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

11. 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

12. So how do we design an electrophoresis chamber? Dye Electrophoresis Commercial versus built box comparisons

13. The Chemistry of Electrophoresis • Electrolysis always occurs during electrophoresis.

14. 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

15. 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

16. R = Electrophoresis system V = Battery tower Physics of Electrophoresis • Simple DC Circuit model • Measure Voltage and Current, calculate Resistance • Determine impact of different buffers on resistance V = IR

17. Direction of current buffer gel Side view of gel box Physics of Electrophoresis • More complex model of resistance – contribution of electrophoresis components on resistance R4 V = Battery tower RTOT R1 R2 R3 RTOT = 1 (1/(R1+R2+R3) + 1/R4)

18. 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?

19. 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 • 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

20. 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

21. Physics of Electrophoresis – Ohmic heating – Calculations • Pave = Vave x Iave • Calculate the average voltage and average current and use these values to calculate the average power • E = Pave x 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 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 Tis thedifference 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 • 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?)

22. 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!!