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Recombinant DNA Techniques chapter 18

Recombinant DNA Techniques chapter 18. Part I. techniques and their applications.  Restriction Digest (to be done in lab) Southern Blot Northern Blot Western Blot Cloning. 1. Restriction Digest: The Cutting of DNA. Endonucleases =. Restricition Enzymes. EXAMPLES OF DNA AGAROSE GELS.

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Recombinant DNA Techniques chapter 18

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  1. Recombinant DNA Techniqueschapter 18 Part I

  2. techniques and their applications. • Restriction Digest (to be done in lab) • Southern Blot • Northern Blot • Western Blot • Cloning

  3. 1. Restriction Digest: The Cutting of DNA Endonucleases = Restricition Enzymes

  4. EXAMPLES OF DNA AGAROSE GELS Ladder, marker or standard of known DNA sizes

  5. An Application of Restriction DigestChromosomal Disorders

  6. Put your thinking cap on!

  7. Wild type Hb Sickle cell Hb Restriction enzyme 1 band is too small to view here) 2 3 Restriction enzyme loss of cutting site

  8. Wt= wildtype M= mutated M M Wt Wt Wt M

  9. An Application to Forensic & Paternity Studies 2. The Southern Blot

  10. 1. Restriction Digest & gel electrophoresis TRANSFER DNA crime scene DNA suspect 2 DNA suspect 1 DNA victim

  11. You must understand the reason for the “probe”!

  12. But there are thousands of fragments!

  13. So sometimes you have to really search for the differences between DNA.

  14. Probe is complementary “hot” DNA

  15. From: Restriction digest To result SUMMARY OF A SOUTHERN BLOT

  16. Suspect 1 Suspect 2 Crime scene Victim Back to our crime scene

  17. Techniques also are available to measure RNA and Protein 3. Northern Blot for RNA 4. Western Blot for Protein

  18. Western Summary: Blotting techniques with probes Southern Northern

  19. 5. Technique: Cloning of specific genes or DNA fragments = restriction enzyme + = ligase

  20. Cut vector Cut foreign DNA or gene Paste together

  21. 2 different antibiotic resistant genes Bacteria are now resistant to 2 antibiotics

  22. Let’s apply these concepts to plasmid transformation.

  23. Cut DNA here & insert foreign DNA

  24. Will your transformed colonies with the insert be white or blue?

  25. Summary of cloning DNA into a blue/white selection vector

  26. End of part 1

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