Difference between revisions of "20.109(S08):Site-directed mutagenesis (Day2)"
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+ | ''First, check out some general [http://openwetware.org/wiki/20.109(S08):_Module_2_General_Comments_on_Lab_Notebooks |comments] on your lab notebooks. | ||
+ | '' | ||
==Introduction== | ==Introduction== | ||
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+ | Last time you navigated a great deal of information in order to design mutagenized inverse pericams nice work! Today you will put your designs into practice. | ||
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+ | [[Image:20.109_SDM-Nobel.png|thumb|right|150px| 1993 Chemistry Nobel Prize co-winner (with Kary Mullis, inventor of PCR) for developing site-directed mutagenesis.]] | ||
+ | As you already know, the mutagenesis strategy you will use shares some features with the PCR you performed in Module 1. You will begin by combining plasmid DNA encoding wild-type inverse pericam with the mutagenic primers you designed. These will be acted upon by a DNA polymerase to generate a mutant plasmid at this stage the plasmid will be nicked, and won t become whole until it is transformed into competent bacteria and repaired by them. In order to propagate only the mutant plasmid, the parental DNA is specifically digested using the DpnI enzyme prior to bacterial transformation. The thermocycling reaction will run for about two hours, and the digestion step for another hour. During these incubation times, we will discuss two articles from the primary literature. | ||
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+ | [[Image:20.109_SG-SDM-P1.png|thumb|left|350px| '''From Stratagene QuikChange® Manual, Part 1''']] | ||
+ | [[Image:20.109_SG-SDM-P2.png|thumb|none|350px| '''From Stratagene QuikChange® Manual, Part 2''']] | ||
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+ | We will warm-up by discussing the paper by [http://www.ncbi.nlm.nih.gov/pubmed/7809066?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Heim, Prasher, and Tsien]. This is a short paper describing the very first attempt to mutagenize GFP, and a fine introduction to some of the concepts and methods used in this module. Next we will do a close reading of the paper that introduced inverse pericam, by [http://www.ncbi.nlm.nih.gov/pubmed/11248055?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Nagai et al]. We will examine the construction and analysis of the IPC multi-component calcium sensor in some depth. | ||
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+ | Now might be a good time to mention why we care about measuring intracellular calcium in the first place. Calcium is involved in many signal transduction cascades, which regulate everything from immune cell activation to muscle contraction, from adhesion to apoptosis - see for example | ||
+ | [http://www.ncbi.nlm.nih.gov/pubmed/18083096?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum this review by David Clapham in Cell], or [http://www.ncbi.nlm.nih.gov/pubmed/11830654?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum this one by Ernesto Carafoli in PNAS]. Intracellular calcium (Ca<sup>2+</sup>) is normally maintained at ~100 nM, orders of magnitude less than the ~mM concentration outside the cell. ATPase pumps act to keep the basal concentration of cytoplasmic calcium low. Often calcium acts as a secondary messenger, i.e., it relays a message from the cell surface to its cytoplasm. For example, a particular ligand may bind a cell surface receptor, causing a flood of calcium ions to be released from the intracellular compartments in which they are usually sequestered. These free ions in turn may promote phosphorylation or other downstream signaling. | ||
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+ | The proteins that bind calcium do so with a great variety of affinities, and have roles ranging from sequestration to sensing. Some calcium responses may have long-term effects, particularly in the case of transcription factors that can bind calcium. As you learned last time, calmodulin works as a calcium sensor by undergoing a conformational change upon calcium binding. Your goal today is to prepare mutant calmodulin (in the context of inverse pericam) DNA, in order to alter the affinity of the resulting protein for calcium. | ||
==Protocols== | ==Protocols== | ||
− | ===Part 1: | + | ===Part 1: Primer preparation=== |
− | #Calculate the amount of water needed ''for each primer'' to give a concentration of 1 | + | #Calculate the amount of water needed ''for each primer'' to give a concentration of 1 mg/mL. |
#Touch-spin your primers, resuspend each in the appropriate volume of water, and touch-spin again. | #Touch-spin your primers, resuspend each in the appropriate volume of water, and touch-spin again. | ||
− | #Calculate the dilution of primer that you need to prepare in order to have 125 ng present in 2.5 | + | #Calculate the dilution of primer that you need to prepare in order to have 125 ng present in 2.5 |