Cloning with Plasmids

1. Cloning with Plasmids

2. 1973 Genetic Engineering Invented

3. MARKER Cloning: Digestion TARGET GENE SOURCE DNA

4. MARKER Cloning: Digested Fragments TARGET GENE

5. MARKER Cloning: Ligation DNA LIGASE TARGET GENE

6. MARKER Cloning: Recombinant Plasmid TARGET GENE

7. Cloning: Transformation E. coli

8. Cloning: Competence Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ E. coli Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+

9. Cloning: Preparing Competent Cells • inoculate culture with a 1/100 dilution of an overnight • grow to an OD600 of 0.2 • transfer to 50 ml Falcon tubes, chill on ice 10'. • pellet in Beckman kneewell centrifuge, 3K, 10'. • resuspend in 12.5 ml 100mM MgCl2 (This is best done by initially resuspending the cells in 1 ml, using a P1000 and then adding an additional 11.5 ml.) • pellet • resuspend in 25ml 100mM CaCl2 (1ml then 24). • incubate on ice for 20 minutes • pellet • resuspend in 0.5 ml 85mM CaCl2 and 15% glycerol • snap freeze in liquid nitrogen, keep @ -70°C.

10. Cloning: Competence E. coli

11. Cloning: Transformation E. coli

12. Cloning: Transformation E. coli

13. Cloning: Transformation After ligations have gone for one hour, you will do the following in order to transform the competent cells: 1) Thaw competent cells and quickly add 150 µl of these cells to each ligation tube. Mix gently. Leave on ice for 20 minutes. 2) Heat shock cells by placing tubes in a 42°C water bath for 90 seconds. 3) Add 0.8 ml L broth and incubate at 37°C with gentle shaking for 1 hour.

14. plasmid plasmid ligase plasmid kan gene ligase Cloning: Plating amp kan

15. Today’s Activities • Set up ligation • Set up digestions to complete map and for blot (use 2x volume for blots) • Transform ligations into competent cells • Plate transformations onto amp and kan plates