AnnouncementsMethods section tipsMethods section exercisesDNA Electrophoresis (EP): PrincipleDNA EP: VisualizationDNA EP: Clean-up and SafetyDNA extraction from agarose gelToday in Lab Announcements Lab Quiz (on M1D1 material) Pre‐lab Lecture Writing a Methods Section Gel Electrophoresis DNA purification Today in Lab: M1D2Announcements• Christina will be back by Day 4, is currently contributing from home• Discussion of orientation day quizMethods 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, primersDNA Electrophoresis (EP): PrincipleAgarose gelDNADriving force for separation:DNA moves to because of Separation is according to:__________ DNA moves faster because‐+Agarose and DNA are bothBiological polymers Æ have molecularentanglementsElectrostatic charge, massPhosphate groups‐+SizeShorterEntanglements increase with sizewt. % increases, pore size decreasesDNA EP: VisualizationLoading dye:Ethidium bromide:glycerol Æ sink into wellsxylene cyanol Æ visual tracking dyefluoresces under UV if bound to DNADNA EP: A n a l y s i sDNA ladder: Relationship: L sample500bpif linear DNA ,~500bpstandards of known size(and concentr a t i o n )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 gelSilica resin column[qiagen.com]Note: initial mixture should look yellow, not bluewhy? isolated desired DNA, change buffer1.bindDNA Æ high salt, low pHchaotropic salt disrupts H‐bondDNA sticks to silica column2.keep DNA washelseethanol –precipitates DNA3.eluteDNA Æ low salt, high pHÆ electrostatic repulsion –Si‐O‐‐O‐P‐DNACourtesy 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 OpenCourseWarehttp://ocw.mit.edu 20.109 Laboratory Fundamentals in Biological EngineeringSpring 2010 For information about citing these materials or our Terms of Use, visit:
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