1a. Alkaline Lysis Method
1a. Alkaline Lysis Method
Inoculate a 5 ml culture of LB/amp (50-100 g/ml) with a single bacterial colony. Place tube in 37C shaker overnight. 2. Fill an eppendorf tube with approximately 1.5 ml of the culture and microfuge 1 minute. Remove the supernatant and add another aliquot of culture to the tube. Again, microfuge 1 minute and remove the supernatant. Repeat until the entire 5 ml culture is spun down in one tube. 3. Resuspend pellet in 100 l of solution I: Solution I 25 mM Tris pH 8.0 100 ml: 1 M Tris 25.0 ml 10 mM EDTA pH 8.0 0.5 M EDTA 2.0 ml 50 mM Glucose 1 M Glucose 5.0 ml dH2O 68.0 ml 4. Add 20 l 10 mg/ml lysozyme solution: 1 ml: 0.01 g lysozyme Fill to 1 ml with 0.250 M Tris pH 8.0 Mix well and let the tube stand at room temp 2 minutes. 5. Add 200 l of Solution II: Solution II 1 ml: 50 l 20% SDS 20 l 10 N NaOH 930 l dH2O Mix and ice 5 minutes. 6. Add 150 l Solution III: Solution III 100 ml: (3 M K+, 5 M Acetate) 5 M KOAc glacial acetic acid dH2O 60 ml 11.5 ml 28.5 ml
5 M Potassium acetate 100 ml: 49.075 g potassium acetate Fill to 100 ml with dH2O IV.A.1
7. Vortex gently to form small white clumps. Ice 5 minutes. 8. 9. Microfuge 5 minutes in cold microfuge. Transfer supernatant to new tube. Add 400 l of phenol:chloroform. Vortex and microfuge 2 minutes.
10. Transfer aqueous (upper) phase to a new tube. Add 1 ml room temp EtOH. Mix well and stand at room temp for 2 minutes. 11. Microfuge 5 minutes in cold microfuge. Pour off EtOH and let pellet dry completely. 12. Resuspend pellet in 50 l of TE/ RNase (20 g/ml): 1 ml: 20 l 10 mg/ml RNase 980 l TE 13. Place tube at 37C for 30 minutes. 14. Restriction digest of mini-prep DNA: 10 l DNA (approximately 1 g) 2 l enzyme buffer 1 l enzyme (approx. 10 units) Incubate for 2-12 hours at 37C (for most enzymes--check appropriate temperature before incubation). 15. Prepare a 1% agarose gel with 0.2% EtBr 100 ml: 1 g agarose 100 ml 1xTBE --boil to dissolve agarose-20 l 10 mg/ml EtBr Add 2 l gel loading dye and microfuge the tube approx. 5 seconds. Load 10 l sample.
IV.A.2