Competent Cell Preparation

y LB broth (250 ml) y Peptone-2.5 g y Nacl-2.5 g y Yeast extract-1.25 g Lb amp plates (250 ml) y Lb broth + 2 % agar (5 g) + 250 l Ampicillin (100 mg/ml) 50 ml sterile tubes-6 Sterile 125 ml flask-6 Solution I y 0.1 M CaCl2 Solution II y 0.1 M CaCl2 y 15 % glycerol Sterile 1.5 ml tubes Control AmpR plasmid

y y y y y

y y

Isolation of plasmid from E.coli

Solution I-50 ml o 50 mM glucose o 25 mM Tris-Cl pH-8 o 10 mM EDTA Solution II-50 ml o 0.2 N NaOH o 1 % SDS Solution III-50 ml o 5M potassium acetate (30 ml) o Glacial acetic acid (5.75 ml) o Water (14.25 ml) y Phenol:Chloroform:Isoamyl alcohol (25:24:1) y 100 % Ethanol y 70 % Ethanol y 10 mM Tris pH-7.5 y Sterile test tubes y RNase-25 mg/ml y LB broth

Experiment I: ISOLATION OF PLASMID DNA FROM BACTERIAL CELLS (MINI PREP)

1. Inoculate a single colony in 4 ml of LB broth with selective antibiotic (LB amp broth) and incubate in 37 C for 12-14 hrs (overnight) at 225 rpm. 2. Centrifuge the cells at 13000 rpm for 2 minutes. 3. Resuspend the cells in 200 l of solution I and vortex vigorously. 4. Add 400 l of solution II and gently mix (donot vortex) and incubate in room temperature for 5 min. 5. Add 300 l of solution III and mix it gently followed by incubation in ice for 5 min. 6. Centrifuge at 13000 rpm for 10 minutes at 4 C. 7. To the supernatant add 1 l of RNase (25mg/ml) and keep it for incubation at 37 C for 30 min. 8. After incubation add equal volume of Phenol:Chloroform:Isoamyl alcohol (25:24:1) and mix well. 9. Centrifuge at 13000 rpm for 5 minutes at 4 C. 10. Add two volumes of 100 % ethanol and mix it well, followed by incubation at room temperature for 2 min. 11. Centrifuge at 13000 rpm for 10 minutes at 4 C. 12. Discard the supernatant and resuspend the pellet in 1 ml of 70 % ethanol. 13. Centrifuge at 13000 rpm for 5 minutes at 4 C. 14. Discard the supernatant and air dry the pellet, followed by resuspend the pellet in 35 l of Tris-Cl (pH-7.5).