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Gel Electrophoresis and Southern Blotting

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

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Gel Electrophoresis and Southern Blotting

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  1. Gel Electrophoresis and Southern Blotting Nick Hasle

  2. Gel Electrophoresis • Overall Purpose: gel electrophoresis is used to separate different strands of DNA according to their base pair lengths in a gel matrix

  3. 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

  4. What it all Looks Like Agarose Gel Ethidium Bromide (EtBr) Tray and Buffer Gel, DNA, and EtBr under UV Light

  5. Result Wells Markers • 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 Samples

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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 nitrocellulose membrane • Then, inserting a radioactive probe will cause only the parts of DNA you are interested in to light up Blotting Heavyweight Paper Towels Gel Sponge Nitrocellulose membrane = capillary action Alkaline Solution

  11. How it Works • The alkaline solution denatures the DNA into single strands • The probe is a piece of DNA or RNA that has the exact opposite sequence of the DNA sequence you’re looking for • This way, it will bind only to the single DNA strands that you want to see

  12. Result • When a piece radioactive-sensitive photographic film is exposed to the nitrocellulose membrane, the radioactive probe will imprint upon it bands similar to those of gel electrophoresis • These bands tell you how far the DNA sequences you were looking for traveled

  13. Biological Questions and Answers & Uses in the Lab • Can be used to find the number of gene copies (or sequence copies) in a single genome • Can differentiate between mutated genes and non-mutated ones if there is a difference in restriction sites for restriction enzymes • E.g. sickle cell anemia

  14. Information http://en.wikipedia.org/wiki/Southern_blot http://en.wikipedia.org/wiki/Probe_hybridization Our wonderful biology book Images http://en.wikipedia.org/wiki/DNA http://en.wikipedia.org/wiki/Image:Agarosegel.jpg http://en.wikipedia.org/wiki/Image:Gel_electrophoresis_apparatus.JPG http://www.gbiosciences.com/ResearchUploads/ResearchProductsImages/mediumimages/GlowDyeGlows-1.jpg http://en.wikipedia.org/wiki/Image:EtBr2.JPG http://en.wikipedia.org/wiki/Image:AgarosegelUV.jpg http://www.babble.com/CS/blogs/famecrawler/2007/08/23-End/question-mark.jpg http://socsci2.ucsd.edu/~aronatas/project/cartoon.doctor.gif http://www.vysis.com/images/content/DNA_Probe.gif http://en.wikipedia.org/wiki/Image:Sicklecells.jpg Bibliography

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