This document summarizes the use of agarose gel electrophoresis to separate DNA fragments by size. Agarose gels can separate DNA fragments up to 20kb in length, with resolution dependent on agarose concentration. DNA fragments are negatively charged and migrate through the gel towards the positive electrode. The separated fragments can be visualized under UV light after staining with ethidium bromide. The mobility of DNA fragments depends on factors like size, shape (linear vs circular), agarose concentration, applied voltage, electrophoresis buffer composition.

1. Experiment 12 Agarose Gel Electrophoresis

2. Separation of DNA Fragments by Gel Electrophoresis Two Types Polyacrylamide gels Agarose gels

3. Polyacrylamide Gels Separate DNA fragments up to 1000 bp long Higher resolving power than agarose Separate fragments that differ in length by single nucleotide

4. Agarose Gels Separate mixtures of fragments up to 20 kb Easier to prepare Lower resolution

5. Agarose Gel Electrophoresis Carbohydrate polymer purified from salt water algae Copolymer of mannose and galactose Melting and cooling form a gel with various pore sizes Overall speed based on concentration of agarose Pulsed-field gel electrophoresis Migration of DNA Negatively charged phosphate backbone size of DNA fragments Size of DNA fragments compared to standard

6. Bands of gel are visualized with UV light source central dye Ethidium Bromide Fluorescent under UV light

8. Results and Discussion Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight.

9. Results and Discussion Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass.

10. Results and Discussion Several additional factors have important effects on the mobility of DNA fragments in agarose gels

11. Results and Discussion Agarose Concentration Higher concentrations of agarose facilitate separation of small DNAs, Low agarose concentrations allow resolution of larger DNAs.

12. Results and Discussion Larger fragments are much better resolved in the 0.7% gel hile the small fragments separated best in 1.5% agarose

13. Results and Discussion Voltage As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments

14. Results and Discussion Electrophoresis Buffer Buffers not only establish a pH, but provide ions to support conductivity. DNA fragments will migrate at different rates due to differences in ionic strength.

15. Results and Discussion Effects of Ethidium Bromide Binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility