BY 260 General Microbiology LaboratoryII Prof Dr Sevgi

BİY 260 General Microbiology Laboratory-II Prof. Dr. Sevgi ERTUĞRUL KARATAY

Isolation of plasmid DNA by alkaline lysis method Plasmids are extrachromosomal DNA structures found in bacteria and some yeasts, which do not encode vital genes, but which can help the survival of microorganisms (antibiotic resistance, some enzymes, etc. ). Plasmids are very important in molecular biology because they are frequently used in cloning studies.

• The basis of the alkaline lysis method is that the p. H changes in the environment have a different effect on chromosomal and plasmid origin DNA fragments. • Basically, when the medium becomes alkali, both chromosomal and plasmid DNA become a single strand. However, when the p. H of the medium is lowered and acidified again, the strands of the small plasmid DNA can merge rapidly, but since the chromosomal DNA is larger, the double-chain formation does not occur easily and the chromosomal DNA is denatured. • In this way, chromosomal DNA and plasmid DNA can be separated.

• Procedure • Requirements • Overnight bacterial culture (E. coli DH 5α) • Sterile Eppendorf Tube • Sterile micropipette tip • Tube holder • Automatic pipette • Microfuges

• a. Generation of appropriate bacterial strains • 1. A single bacterial colony is grown in LB (Luria Bertani) medium containing 2 ml of appropriate antibiotics in a shaking oven at 37 ° C overnight. • 2. Transfer 1. 5 ml culture to an Eppendorf tube and centrifuge for 30 sec at 13000 rpm. • 3. The supernatant is discarded and the pellet is used for plasmid isolation.

• b. Plasmid isolation by alkaline lysis method • 1. The bacterial pellet is diluted with 200 µL of cold solution I and vortexed. • 2. Add 200 µl of solution II (160 ml Na. OH + 40 ml SDS). The tube is turned upside down and mixed well 5 times. Leave on ice 10 '. • 3. Add 250 µl of cold solution III. Vortex and stand on ice for 15 '. • 4. Centrifuge 5 'at 13000 rpm. The supernatant is transferred to a clean tube. • 5. Add an equal volume of phenol: chloroform, vortex.

• b. Plasmid isolation by alkaline lysis method • 6. Centrifuge at 2 min for 13000 rpm. The supernatant is taken to a clean tube. • 7. Add two volumes of pure alcohol to the supernatant and allow to stand on ice for 15'. • 8. Plasmid DNA is precipitated by centrifugation at 13000 rpm for 5'. • 9. The alcohol on the pellet is thoroughly dried and 70% cold ethanol is added and the pellet is washed. • 10. Remove the alcohol and dissolve the pellet in 10 ml of sterile distilled water.

• Preparation of solutions: • Solution I: • 50 m. M Glucose • 25 m. M Tris. HCl (p. H: 8. 0) • 10 m. M EDTA (p. H: 8. 0) • Solution II: • 0. 2 N Na. OH • % 1 SDS • Solution III: • 5 M potassium acetate 60 ml • Glacial acetic acid 11. 5 ml • H 2 O 28. 5 ml

• EDTA disrupts the cell structure by binding Mg ++ ions which are necessary for the protection of the cell structure and inhibits the cell enzymes that enable the degradation of DNA. • SDS removes lipid molecules by lysis and disrupts the cell membrane structure. • Phenol-chloroform precipitates the proteins and removes them from the DNA. • Pure Ethanol competes with the H 2 O molecules in the medium to allow DNA to collapse. • 70% Ethanol, Na+, such as monovalent cations and salts to dissolve away from the environment.