Objective

Preparing Lambda DNA from Phage Lysates.

Overview

A scaled-up liquid lysate is used to prepare a large quantity of highly purified phage DNA. Phage is separated from cellular debris by a CsCl gradient centrifugation.

Reagents required

1. 10 mM Tris (pH 7.4)
2. 10 mM MgCl₂
3. Chloroform
4. 0.2 M NaCl
5. Absolute ethanol
6. 70% ethanol
7. TE Buffer
8. TM Buffer
9. 10% Poly Ethylene Glycol (PEG)
10. CsCl (0.72 g/ml)

Equipments Required

1. GSA rotor
2. Erlenmeyer flask
3. Pasteur pipette
4. Serological pipette
5. Polyallomer centrifuge tube
6. Centrifuge - Ti50 (Beckman) or equivalent
7. 22-gauge needle fitted in a plastic syringe
8. HB-4 type rotor centrifuge

3 Days

Procedure

1. Extract the phage DNA

1. A confluent plaque is covered with 4 ml of 10 mM Tris, (pH 7.4), 10 mM MgCl₂, lambda buffer (TM), is gently shaken and maintained in the refrigerator for two hours.
2. Add 3ml of chloroform, the plate is manually shaken, slightly slanted and the buffer withdrawn with a Pasteur pipette.
3. One ml of the phage suspension is mixed with 1 ml of a competent overnight culture resuspended in 10 mM MgCl₂ in 0.4 volumes of the original LB medium
4. Keep the tube at 37^oC for 20 minutes and used to inoculate 400 ml of LB, 10 mM MgCl₂, in an Erlenmeyer flask.
5. Following an overnight incubation at 37^oC, under strong agitation, using only a piece of aluminum foil attached with tape to the mouth of the flask for better aeration; tread like indicates that the material has been obtained in good yield.
6. Add 2 ml of chloroform, mix the suspension vigorously, and then made up to contain 50 g/l of NaCl.
7. After dissolution of the salt, the suspension is centrifuged for 10 min at 8,000 rpm in a type GSA rotor, to pellet cells and debris.
8. The supernatant is transferred to an Erlenmeyer flask containing 40 g (10%) PEG, kept on ice for at least one hour. The suspension is then agitated, centrifuged for 10 min at 8,000 rpm, and the supernatant

2. CsCl centrifugation

1. The precipitate on the tube wall is resuspended in approximately 5 ml of lambda buffer with the help of a serological pipette, and transferred to a polyallomer centrifuge tube, previously marked to 9 ml, and containing 6.48 g of CsCl (0.72 g/ml).
2. The volume is made up to 9 ml with lambda buffer and dissolve the salt.
3. The tube is filled with mineral oil (or resuspension medium), and is centrifuged overnight in a fixed angle rotor (Ti50 (Beckman) or equivalent) centrifuge at 45 K.
4. After the run, a bluish white band, formed which is removed after puncturing the tube with a 22-gauge needle fitted in a plastic syringe.
5. The phage suspension is collected in a tube containing 2 ml of 10 mM Tris, pH 8.0, EDTA 1 mM (TE), which is transferred to a dialysis bag and the suspension dialyzed for two hours against TE.

3. Phenol extraction of DNA samples

1. The content of the dialysis bag is transferred to an appropriate centrifuge tube, TE is added to the tube until the volume is raised to approximately 5 ml and extracted for 10 min by inversions with an equal volume of Tris buffer (pH 8.0) and saturated phenol, containing 0.1% 8-hydroxyquinoline
2. Centrifuge the mixture for 1o minutes at 8000 rpm in an HB-4 type rotor centrifuge.
3. After Centrifugation the aqueous phase is carefully removed, made up to 0.2 M NaCl and precipitated by gentle addition of cold absolute ethanol
4. The rod with DNA is briefly dipped in a tube containing 70% ethanol in order to become free from traces of phenol.
5. Touch in the border of this tube to discard excess of ethanol and, without drying, the DNA is dissolved and maintained in 0.5 or l.0 ml of TE.

4. Notes

During Phenol Extraction Gloves should be worn to prevent severe phenol burning.

5. Recipes

TE Buffer

10 mM Tris-Cl, pH 7.5

1 mM EDTA

TM buffer

50 mM Tris-Cl, pH 7.5

10 mM MgSO₄

Products Available

1. MgCl2
2. TE Buffer
3. TM Buffer
4. Tris
5. Poly Ethylene Glycol (PEG)
6. CsCl

References

1. Kaiser K, Murray NE, Wittaker P, Construction of representative genomic DNA libraries using phage lambda replacement vectors. In: DNA Cloning 1 - A Practical Approach (Glover, D.M. and Hames, B.D., eds.). Chapter 2. IRL-Press, Oxford University Press, New York, pp. 1995: 37-82.
2. Kirby KS A new method for the isolation of deoxyribonucleic acid: Evidence on the nature of bonds between deoxyribonucleic acid and protein. Biochem. J. 1957: 66: 495-504.
3. Latchman DS, Brickell, P, An improved method for the isolation of high yields of bacteriophage lambda DNA. Nucleic Acids Res. 1986: 14: 5220.
4. Schleif RF, Wensink, P, Lambda DNA purification. In: Practical Methods in Molecular Biology (Manor, E.P., ed.). Springer-Verlag, New York. 1981