Gel Electrophoresis and Southern Blotting凝胶电泳和Southern印迹.ppt

Gel Electrophoresis and Southern Blotting Nick Hasle Gel Electrophoresis Overall Purpose: gel electrophoresis is used to separate different strands of DNA according to their base pair lengths in a gel matrix How it Works DNA has phosphate groups that are negatively charged When DNA that’s suspended in a gel medium is affected by an electrical current, it will travel towards a positively charged electrode Larger pieces of DNA move more slowly through the gel than smaller pieces What it all Looks Like Agarose Gel Tray and Buffer Ethidium Bromide (EtBr) Gel, DNA, and EtBr under UV Light Result When you “run” a DNA sample through a gel, DNA strands with the same number of bases (i.e. those of the same length) will separate into “bands” along the gel Markers can be used to estimate the length of DNA strands in any particular band Markers Samples Wells Uses in the Lab Can be used as a visual verification that another procedure (PCR, mini-prep, etc.) has been correctly completed E.g. testing whether a restriction enzyme works or not Uses in the Lab Can be used to further purify a sample of DNA After the DNA samples are run through the gel, you can cut the gel in the places where the desired DNA sequence lies, as long as you know its length Then, you can run a gel extraction protocol to isolate the purified DNA sample Cut out these bands to only get DNA of a specific length Biological Questions and Answers Can compare/analyze the length of DNA (obviously) Can determine whether or not an individual has a particular allele of a certain gene E.g. sickle cell anemia Can check the effectiveness of a novel biological technique Southern Blotting Overall Purpose: to separate a certain piece of DNA (of which you know the sequence) from a DNA sample E.g. a particular gene from an entire genome How it Works Uses a combination of gel electrophoresis, capillary action, and nucleic acid hybridization After a sample of DNA is run through a gel, you can blot the sample onto a nitrocellul