Review Session

1. You will be given a strand of wild type DNA and be ask to transcribe and translate it. Also you will be given 2 mutant DNA strands which you will have to transcribe and translate. Then you will need to know which mutation accounts for a certain phenotypic change and explain (you will be given a scenario).
2. Techniques you need to know: Transformation, Western Blot, Pull down Assay, PCR, Site-Directed mutagenesis (Casizzle)
3. Definately know what PCR is…you will be given a scenario based on PCR and what gene is amplified (say the gene PCR is amplifying is 1000 bp) then after running electrophoresis, a gel with bands will appear. If there isnt a band at the 1000 bp area, then something went wrong…you need to come up with some reason why there is not a band at 1000 bp (also why there might be bands in different locations) and explain why. Example, say one band ended up at the way bottom of the gel (meaning it was a very small gene/sequence of bases) but no band at 1000bp. One reason for this would be that the primers used in PCR were not complimentary to the gene being amplified, therefore the primers paired up with eachother, and that accounts for the band seen at the bottom of the gel.
4. Also Definately know what Site-directed mutagenesis is. You will be given a wt amino acid sequence in which you hypothesize that one amino acid is responsible for binding to a particular protein. To test this, run S-d mutagenesis which involves PCR that uses a primer in which one base pair does not complimentrary bind to one base pair that codes to that particular amino acid responsible for protein binding. Run Pcr.
5. Pull down assay-involves attaching a certain amino acid sequence to agarose gel beed and introducing this complex to a particular protein you believe it binds to, centrifuging it, boil off agarose, and running western blot with primary antibody against that particular protein.(i might go in more detail lata…just too tired right now) I'd like to point out here Little Beau Peep that it just isn't agarose beads and boiling. There are several different techniques that can be used including using DIFFERENT beads. You also don't have to boil the beads, often using a high concentration salt buffer can break the bond between the bead and the fusion protein, or chemically modified protein. You can then run a Western Blot with the supernatant without having to boil off your beads.
6. Know main concept behind why bull terriers have sloped faces
7. Also know meselson-stahl exp

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