Announcements Lab Quiz on M1D1 material Pre lab Lecture Writing a Methods Section Gel Electrophoresis DNA purification Today in Lab M1D2 Announcements Christina will be back by Day 4 is currently contributing from home Discussion of orientation day quiz Methods section tips Organizing sub sections Start with an overview sentence then detailed steps Methods should be concise and complete Space wise avoid tables lists if a sentence suffices Sentence wise avoid extra confusing words Content wise cover what s needed and only that which is needed to understand and replicate your experiments Concentrations are more useful than volumes or you can state amounts plus total volume Methods section exercises Which is more readable To the Y were added the X or The X were added to the Y How can I more quickly express 1 g of protein in 45 mL of water and 5 mL of 10X buffer B 2 protein in aqueous buffer B Which parts of a PCR are unique to a given experiment versus standard protocol Tanneal textension 1min kbp plasmid cycles composition concentration of template primers DNA Electrophoresis EP Principle Agarose gel DNA Agarose and DNA are both Biological polymers have molecular entanglements Driving force for separation Electrostatic charge mass DNA moves to because of Phosphate groups Separation is according to Size Shorter DNA moves faster because Entanglements increase with size wt increases pore size decreases DNA EP Visualization Loading dye glycerol sink into wells xylene cyanol visual tracking dye Ethidium bromide fluoresces under UV if bound to DNA DNA EP Analysis L sample DNA ladder standards of known size and concentration Relationship 500bp if linear DNA 500bp DNA EP Clean up and Safety Use nitrile gloves when handling DNA gels and all equipment used for gels Gels and gel contaminated papers are disposed of in solid chemical waste Wear eye protection face shields when cutting DNA bands out of a gel DNA extraction from agarose gel why isolated desired DNA change buffer 1 bind DNA high salt low pH chaotropic salt disrupts H bond DNA sticks to silica column 2 keep DNA wash else ethanol precipitates DNA Silica resin column qiagen com 3 elute DNA low salt high pH electrostatic repulsion Si O O P DNA Note initial mixture should look yellow not blue Courtesy of QIAGEN GmbH Used with permission Today in Lab Set up gel runs 60 min we will photograph it Mark your area of the gel box with coloured tape Bring your USB key up front Meanwhile discussion w Neal and Linda Finally DNA extraction from gel FNT methods section read journal article MIT OpenCourseWare http ocw mit edu 20 109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use visit http ocw mit edu terms
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