1 / 20

GEL ELECTROPHORESIS

GEL ELECTROPHORESIS. Gel Electrophoresis. Separation of molecules by size, charge, or shape PAGE ( P oly A crylamide E el E lectrophoresis ) Used to separate proteins Proteins are kept the same shape by detergent (SDS) Agarose Used to separate DNA fragments DNA can be recovered from gel.

farren
Download Presentation

GEL ELECTROPHORESIS

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. GEL ELECTROPHORESIS

  2. Gel Electrophoresis • Separation of molecules by size, charge, or shape • PAGE (PolyAcrylamideEel Electrophoresis) • Used to separate proteins • Proteins are kept the same shape by detergent (SDS) • Agarose • Used to separate DNA fragments • DNA can be recovered from gel

  3. Agarose Gel • Linked dextrose sugars leave channels for the DNA segment to “snake through” • Larger the fragment, the longer it takes for the DNA to travel • Gel is placed in a conductive buffer • Chamber delivers electricity • DNA distance can be gauged by watching loading dye

  4. DNA is negative (PO4 backbone) It runs to the positive electrode The further it moves, the shorter the DNA DNA Gels

  5. How can you adjust GE? • Run gels faster • Higher voltage • Lower temp to keep gels from melting • Does not affect resolution • Make agarose more concentrated • See smaller differences • More concentrated = more separation

  6. Gels • This is an actual restriction digest • You can measure the distance the DNA traveled from the gel • Then you can determine the size of unknown fragments as long as you have a known

  7. Size Determination • Graph the known fragment sizes on semi-log paper • Find the distance the unknown traveled • Then extrapolate to the y axis to find the size

  8. Lambda DNA

  9. Lambda DNA digest

  10. Lambda Restriction Sites • KNOWNS • HindIII: • 23,130 • 9,416 • 6,557 • 4,361 • 2,322 • 2,027 • UNKNOWNS • BamHI: • 16,841 • 7,233 • 6,770 • 6,527 • 5,656 • EcoRI: • 21,226 • 7,421 • 5,804 • 5,643 • 4,878 • 3,530

  11. RFLP • Restriction fragment length polymorphism • Differences in DNA sequence on homologous chromosomes that can result in different patterns of restriction fragment lengths • Based on differences in alleles • On average, 2 humans differ 1 in 1000bp • These 2 people will differ in 3 million bp • Rarely have same exact distance between these restriction sites = polymorphic

  12. Southern Blotting • Used to verify the presence or absence of a specific nucleotide sequence in different DNA samples

  13. Southern Blotting

  14. DNA Fingerprinting • Different peoples’ DNA have different restriction sites • Restriction digests on our DNA cut different places, giving unique banding patterns • This is your DNA fingerprint

  15. DNA Fingerprinting • CSI • Match crime scene RFLPs to suspect RFLPs • The match is the killer • Paternity testing

  16. Figure 20.17 DNA fingerprints from a murder case

More Related