Slide 1

1 Trends in Biotechnology 150401 TB 09 Basic tools of recombinant DNA

Slide 2

2 Endonucleases

Slide 3

3 Cutting double-stranded DNA. The enzyme DNase cuts DNA at random sites.

Slide 4

4

Slide 5

5 Fig. 3.1 (b) Specific site cleavage.

Slide 6

6 Video: Restriction http://www.dnalc.org/resources/animations/re striction.html

Slide 7

7 A specific restriction enzyme cuts each DNA molecular at the same sequence site.

Slide 8

8 The ligation of two different pieces of DNA.

Slide 9

9 DNA ligase is an enzyme which remakes the phosphate diester bonds.

Slide 10

10 Video: restriction enzymes bio37 http://highered.mcgraw- hill.com/olc/dl/120078/bio37.swf Video: restriction

Slide 11

11 Agarose gel electrophoresis is used to separate DNA (and RNA) molecules according to size.

Slide 12

12 Electrophoresis: gelelectrophoresis The pieces of DNA move because of the charge on their phosphate groups. They are separated because of their length. With some methods, they can be separated because of their shape. Tutorial on electrophoresis: http://www.sumanasinc.com/webcontent/anima tions/content/gelelectrophoresis.htmlhttp://www.sumanasinc.com/webcontent/anima tions/content/gelelectrophoresis.html Animation of electrophoresis: http://www.dnalc.org/resources/animations/gelelectrophoresis.htm l

Slide 13

13 Ligation of a DNA fragment into a vector using the restriction endonuclease EcoRI and DNA ligase.

Slide 14

14 The transformation of bacteria and the selection of cells with the recombinant vector.

Slide 15

15 Video: Key Steps of Molecular Cloning http://www.youtube.com/watch?v=sjwNtQYLKeU&fea ture=related Video: Plasmid Cloning http://www.youtube.com/watch?v=acKWdNj936o http://highered.mcgraw- hill.com/sites/0072437316/student_view0/chapter1 6/animations.html#

Slide 16

16 Restriction map of pBR322 showing unique restriction sites, the ampicillin and tetracycline resistance genes, and the origin of replication.

Slide 17

17 Note how the cutting and insertion at a particular restriction site will result in loss of resistance.

Slide 18

18 Selection for recombinant cells after transformation.

Slide 19

19 Selection for recombinant cells after transformation.

Slide 20

20 Selection for recombinant cells after transformation.

Slide 21

21 Replica plating

Slide 22

22 Restriction map of pUC18/pUC19 showing the ampicillin resistance gene, the origin of replication, and the multiple cloning site within the lacZ gene.

Slide 23

23 The selection of bacterial colonies containing a recombinant vector (pCU vector + DNA insert) by the selection of white colonies.

Slide 24

24 Cloning using Bacteriophage The lytic life cycle of a bacteriophage.

Slide 25

25 Fig. 3.11 The cloning of human DNA restriction fragments ( and ) into a bacteriophage vector.

Slide 26

26 Fig. 3.12 The use of Agrobacterium tumefaciens to transfer DNA into plants.

Slide 27

27 Video: Ti plasmid http://highered.mcgraw- hill.com/sites/0072437316/student_view0/ch apter16/animations.html#

Slide 28

28 Fig. 3.13 Particle gun used for transforming cells with DNA-coated microprojectiles.

Slide 29

29 An animation of a gene gun can be found at: http://www.hort.purdue.edu/hort/courses/H ORT250/animations/Gene%20Gun%20Animat ion/Genegun1.html http://www.hort.purdue.edu/hort/courses/H ORT250/animations/Gene%20Gun%20Animat ion/Genegun1.html

Slide 30

30 Fig. 3.14 Microinjection of DNA into pronucleus of a fertilized animal egg.