1. Lecturer: David

3. * Reverse transcription PCR * Used to detect RNA levels * RNA is converted to cDNA by reverse transcriptase * Then it is amplified similar to PCR

4. * This technique can detect very low numbers of RNA * Useful in the insertion of genes into prokaryotes * cDNA is reverse transcribed from mRNA, so no introns are found on the DNA sequence only exons * No splicing is required

5. * Amplification of DNA * Using reverse transcriptase to convert RNA  cDNA * Amplification of cDNA

6. * Allows us to know whether the correct DNA fragments were generated. * Separates fragments by size

7. * Southern Blot * Northern Blot * Western Blot * ELISA

8. * Used to detect a specific DNA sequence * First: Restriction endonucleases are used to cut DNA into fragments * Then: They are run on agarose electrophoresis to separate by size * Next: A sheet of nitrocellulose paper is placed over the gel and baked in vacuum or oven

9. * You expose the membrane to a hybridization probe – a specific DNA sequence * Hybridization probe – a DNA or RNA fragment used to detect a complementary DNA sequence * DNA binds to the membrane since DNA is negatively charged, and the membrane is positively charged * The probe is labeled radioactively or with a fluorescent dye * Any excess probe is washed off and the pattern is then visualized via x-ray

10. * Hybridization of the probe to the DNA fragments indicates the fragment contains a complementary sequence to the probe.

11. * Technique used to study gene expression by detecting RNA * RNA is extracted from a tissue or cells * The RNA sample is then separated by size through gel electrophoresis * A nylon membrane is placed over the gel and RNA is transferred from gel to membrane * The nylon membrane is positively charged which binds effectively to the negatively charged RNA

12. * Once the RNA is bound to the membrane it is covalently linked to it via UV light or heat * A tagged probe is then hybridized with the RNA * This technique allows you to observe gene expression

13. * This technique is used to detect protein samples in cells * Cell samples are broken down mechanically by a blender or lysed then precipitated. * The samples are run on gel electrophoresis and may be separated by: * Isoelectric point – the pH when the protein as no charge * Molecular weight * charge

14. * The most common gel electrophoresis used for proteins is polyacrylamide gel with sodium dodecyl sulfate * SDS-PAGE for short * SDS-PAGE denatures the proteins and allows for separation based solely on molecular weight * SDS coats the protein with a negative charge and proteins travel to the positive anode.

15. * The proteins on the gel are then transferred to nitrocellulose paper by using an electric current * Primary antibodies are mixed with the membrane and binds to the protein * Secondary antibodies, which are tagged, are added and bind to the primary antibodies

16. * Enzyme-Linked ImmunoSorbent Assay * A test that uses antibodies and color changes to identify a substance * An unknown amount of antigen is affixed to a substrate * Then, a specific antibody is washed over the surface so that it can bind to the antigen

17. * Antibody is linked to an enzyme * Substance is added that the enzyme can convert to some detectable signal

18. * Sample with unknown amount of antigens is fixed to medium (plate/well, etc) * Detection antibody is then added, forming a complex with the antigen * Detection antibody can be covalently linked to an enzyme, or a secondary antibody attached to an enzyme can be used

19. * Between each step the plate is typically washed with a mild detergent solution * Detergent removes any proteins or antibodies that are not specifically bound * The plate is developed by adding an enzymatic substrate to produce a visible signal * Signal indicates the quantity of antigen in the sample