Scribd
Upload
35 views2 pages

1a. Alkaline Lysis Method

This document provides instructions for an alkaline lysis mini prep procedure to isolate plasmid DNA from bacterial cells. The procedure involves growing bacterial cells overnight, lysing the cells using solutions containing lysozyme, SDS, and potassium acetate, precipitating the DNA with ethanol, and resuspending the DNA pellet in TE buffer containing RNase for storage and downstream applications like restriction enzyme digestion.

Uploaded by

Parijat Banerjee
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
35 views2 pages

1a. Alkaline Lysis Method

This document provides instructions for an alkaline lysis mini prep procedure to isolate plasmid DNA from bacterial cells. The procedure involves growing bacterial cells overnight, lysing the cells using solutions containing lysozyme, SDS, and potassium acetate, precipitating the DNA with ethanol, and resuspending the DNA pellet in TE buffer containing RNase for storage and downstream applications like restriction enzyme digestion.

Uploaded by

Parijat Banerjee
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 2

ALKALINE LYSIS MINI PREP PROCEDURE (Procedure from Maniatis cloning manual) 1.

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

You might also like