1. Electrophoresis 7 th Grade,

2. Learning Objectives Understanding how electrophoresis facilitates the separation of molecules Be familiar with the types of electrophoresis gels and their uses Understand the process of running an agarose gel Know the conditions and supplies necessary to visualize and interpret a gel Understand the importance of DNA markers

3. What is Electrophoresis? This technique uses electrical currents to separate different sized molecules. Electrophoresis gel is porous so that molecules can move through it.

4. Separation of Molecules by Size Small molecules can navigate through the pores of the gel more easily than large molecules. Small molecules migrate farther in the gel than large molecules.

5. Types of Gels Agarose Best for separating large molecules ex. DNA or RNA greater than 100 base pairs Gel usually runs horizontally Polyacrylamide Best for separating smaller molecules ex. Proteins, DNA and RNA less than 100 base pairs Gel runs vertically

6. Agarose vs. Polyacrylamide Gels AgarosePolyacrylamide Horizontal or vertical? HorizontalVertical Big gaps or small gaps in gel? Bigger gaps than with polyacrylamide Smaller gaps Separates large molecules or small ones? Both, but better for large molecules Both, but better for small molecules Lets you tell small differences between sizes of bands? NoYes Type of molecules run in this type of gel Mostly DNADNA or proteins

7. Use of Agarose Gels Quantify DNA or RNA Isolate fragments for purification Identify fragments based on size Ex. Once DNA is digested by restriction enzymes, you can use electrophoresis to confirm that your digestion was successful

8. Use of Polyacrylamide Gels Determining protein size Determining sample purity Quantifying proteins (protein concentration) Protein identification Western Blot A technique use to determine a specific protein

9. Western Blot - Transferring After protein separation by electrophoresis, protein is transferred to a membrane The membrane is typically nitrocellulose Transfer occurs by placing the membrane on top of the gel, stacking filter paper on top of that, and placing the stack in a buffer solution

10. Western Blot Mechanism Buffer solution moves up the paper by capillary action Protein travels with the buffer solution This leads to the transfer of the protein onto the nitrocellulose membrane Membrane is probed by antibodies for specific protein

11. Western Blot – Antibody Probes Antibodies will only join to their antigen, the molecule that they are specifically made to bind to. This allows for specific protein identification.

12. Making and Loading a Gel Casting a Gel Agarose Solution Casting Tray Loading a Gel Fill the chamber with a running buffer, usually with Tris/Borate/EDTA (TBE) Buffer TBE conducts current through the gel Maintains the pH of the gel to protect DNA from degradation

13. Loading Samples into Gels Loading dye is denser than water, therefore when added to sample, it causes the DNA to sink into wells Allows one to see that the sample has been loaded properly

14. Running a Gel Current runs from the negative electrode (anode) to the positive electrode (cathode). DNA is negatively charged and will migrate down the gel to the positive charge Small fragments will migrate faster and further than large fragments DNA fragments of similar lengths will group together

15. Interpreting Gels DNA appears as bands on the gel Stains like ethidium bromide and Carolina Blue bind to DNA so that it can be seen under light EtBr: Visible under UV light CB: Visible under fluorescent light

16. The DNA Ladder The DNA ladder is a standard that contains fragments of known lengths and concentrations The ladder is run next to your sample It is used to determine the size of DNA in your samples

17. DNA Ladder Sizes DNA is made up of nucleotides, each of which contains a nitrogenous base DNA is measured in base pairs (bp) and kilobases (Kb) 1kb= 1000 bp The DNA Ladder can indicate the length of base pairs in a particular DNA fragment Longer (and heavier) fragments are found at the top of the ladder Shorter (and lighter) fragments are found at the bottom of the ladder

18. 4 Nitrogenous Bases: Purines Adenine (A) Guanine (G) Pyrimidines Thymine (T) Cytosine (C) Phosphate group Deoxyribose (sugar) A binds to T (2 H- bonds) C binds to G (3 H- bonds) DNA Bases

19. Today in Lab Beginning of a two day laboratory DNA Restriction Digest Practical application of almost everything seen in this lecture!

20. Virtual lab http://learn.genetics.utah.edu/content/labs/gel/