DNA Extraction and Amplification University of RochesterDNA提取和扩罗切斯特大学.pptVIP

George Wolfe Loudoun County Public Schools Academy of Science GWOLFE1@LOUDOUN.GOV DNA Extraction and Amplification EXTRACTION ISOLATION OF DNA FROM INSECT SAMPLE ELIMINATION OF CELLULAR DEBRIS ELUTION OF PURIFIED DNA STEP ONE OVERVIEW:ISOLATION Cell Lysis Solution-destroys cell membranes Proteinase K-Destroys DNAses Protein Precipitating Solution precipitates Protein (duh)! At this point you have a “soup” of cellular components. The DNA must now be removed. STEP TWO-ELIMINATION OF CELLULAR DEBRIS Cell “soup” is added to a spin column. A filter in the column attracts DNA, Proteins, and other cell components. A series of buffers/centrifugations will wash out everything but the DNA. Step 3-DNA Elution The DNA is still stuck to the original filter in the spin column. Everything else should be gone. A final Elution Buffer is added, the sample is centrifuged, this removes the DNA. Places where your students (but certainly not you) will mess this up? Losing track of what you have or have not added (see organization chart) Not labeling tubes properly. Waiting too long to add Proteinase K Not changing pipette tips and macerators Throwing out their eluted DNA (yes, this is a common mistake!) DNA AMPLIFICATION- PCR Eluted or Control DNA (+ and -) Master Mix Taq Polymerase Buffers DNTP’s MgCl2 Primers-Forward and Reverse (W spec) More places for your students (but not you) to mess up! Tube Labeling Keeping track of what has been added. We are using “pelleted” master mix See organizational chart * * *