1. Recombinant DNA Techniques chapter 18 begins page 510
Part I

2. We’ll discuss a number of techniques and their applications.
Restriction digest
Southern Blot
Northern Blot
Western Blot
Cloning

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

6. An example of cohesive or staggered cuts

7. Number of fragments vary between linear and circular DNA

8. How do you view the DNA fragments?

9. Bio 22 student
DNA

11. EXAMPLES OF DNA AGAROSE GELS

12. An Application of Restriction Digest Chromosomal Disorders

13. Put your thinking cap on!

14. 2 3 Wild type Hb Sickle cell Hb Restriction enzyme Restriction enzyme
1 band is too small to view here) 2 3
Restriction enzyme
loss of cutting site

15. Wt= wildtype
M= mutated
M M
Wt Wt
Wt M

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

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

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

19. But there are thousands of fragments!

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

21. Probe is complementary “hot” DNA

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

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

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

25. Summary: Blotting techniques with probes
Western
Southern
Northern

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

27. Cut vector
Cut foreign DNA or gene
Paste together

28. 2 different antibiotic resistant genes
Bacteria are now resistant to 2 antibiotics

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

30. Cut DNA here &
insert foreign DNA

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

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

33. End of part 1