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Lecture3Spr08

Lecture3Spr08 - Announcements*Wednesday 15 minute Quiz 8:40...

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Unformatted text preview: Announcements *Wednesday 15 minute Quiz: 8:40 - 8:55. ENDS 8:55 SHARP! Covers material in Lectures 1 - 3. Reminder: six biweekly quizzes in course, can drop one, each counts 2% of course grade. In the future, the Wednesday quiz will cover the previous four lectures, i.e. back to the last quiz. *This Friday there will be an “Enrichment” presentation. 9:05 - 9:55, 245 Warren. See Blackboard for details. Senior grad student Ryan Lewis (Hess lab) will talk on “What's so 'exciting' about membrane permeability?” *Assigned reading for lectures 3 + 4 are now in the Assignments Folder on Blackboard. *“SKIM” means you should be familiar with the basic concepts, but do not need to know the details. *For the quizzes as well as the three exams, most of the questions come out of the lectures, but a few will be from the textbook material that has not been covered in detail in the lectures. Review : Transfection Microinjection Electroporation “Chemical” transfection Old Alberts *Called “TRANSFECTION ( term = “transformation” in E. coli or yeast). *Transfection ef¡ciency varies from >50% of cells taking up DNA to <1%, depending on cell type and method. *Transfection is largely empirical; mechanism poorly understood. *For “chemical” (right), aggregating DNA with polycations or positively charged lipids is most common; DNA does not have to be ‘inside ’ a vesicle as shown. *Called “transient transfection” if cells are assayed within a few days. Desired gene is expressed but expression decays over time because the DNA is not replicated (diluted out as cell divides). *Cells that successfully take up DNA take up a lot (many copies). This is the basis of “co-transfection”: If DNA1 and DNA2 are transfected together and 10% of the cells take up DNA1, most (> ~ 90%) of these cells also take up DNA2. *Called “stable transfection” if DNA is integrated into a chromosome and therefore permanent. *Only a small fraction of the cells that take up DNA transiently integrate the DNA (at random places in chromosomes). Thus, such cells must be selected . Standard way is to co-transfect or otherwise co-express a selectable marker gene. * Example: the drug puromycin kills cells, but if a cell expresses the puro- resistance gene (from bacteria), it will survive and continue to grow. Review : Transfection Review : Radioactive labeling of macromolecules in cultured cells on a plate *Macromolecules can be speciFcally labeled by feeding cells radioactive precursors: amino acids for proteins, nucleosides or bases for RNA (typically uridine) or DNA (typically thymidine). *Typically cellular proteins are labeled with 35S-methionine, because this is cheap, has high speciFc activity (because the half life is short = 3 months, as opposed to 12 yrs for 3H or 5000 yrs for 14C), and speciFc (Met and Cys = only AAs that have sulfur, none in RNA or DNA)....
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Lecture3Spr08 - Announcements*Wednesday 15 minute Quiz 8:40...

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