Molecular Techniques and Methods

Lamda Phage DNA Vector Preparation
for Library Construction

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Alkaline Phosphatase 10 x Buffer (1 ml)
500 mM Tris-HCl (pH 9.0) ---------------------- 500 ul of 1 M Tris-HCl
10 mM MgCl2 ----------------------------------- 10 ul of 1 M MgCl2
l mM ZnCl2 -------------------------------------- 1 ul of 1 M ZnCl2
l0 mM Spermidine ------------------------------- 10 ul of 1 M Spermidine
Distilled H2O ------------------------------------ 479 ul




PROCEDURES

Restriction Enzyme Digestion of lamda DNA Vector Arms

1. Prepare lamda DNA by plate lysis method or liquid culture method.

2. Digest 10-50 ug of lamda phage vector DNA with a 4- to 5-foldexcess of the restriction enzyme for 2 hours.

3. Check that the reaction has gone to completion by electrophoresis of a sample on an agarose minigel.

Sample Enzyme Reaction (250 ul)

Lambda vector DNA (0.5 ug/ul)
25 ul
10 X Buffer
25 ul
Acetylated BSA (1 mg/ml)
25 ul
Restriction Enzyme (20u/ul)
3 ul
Distilled H2O
172 ul

  • Incubate at 37oC for 2 hours.


    Dephosphorylation of Vector Arms

  • This step is optional, depending on the cloning strategy being utilized.
  • Dephosphorylating the ends of the vector will further reduce the background of nonrecombinant plaques obtained.

    4. Sample Reaction (400 ul)

    Restriction enzyme-digested lambda vector DNA from step 3
    250 ul
    10 x Buffer of Alkaline Phosphatase
    40 ul
    Calf Intestinal Alkaline Phosphatase
    20 ul
    Add distilled H2O to make a final volume of
    400 ul

  • Incubate for 1 hour at 37oC.


    5. Extract twice with 1 volume of TE-saturated phenol: chloroform. Vortex gently and centrifuge at 12,000g for 5 minutes.

    6. Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform: isoamyl alcohol (24:1).
    Vortex for 1 minute and centrifuge at 12,000g for 5 minutes.

    7. Transfer the upper, aqueous phase to a fresh tube.
    Add 0.5 volume of 7.5M Ammonium acetate. Add 2 volumes of 100% Ethanol and leave at -70oC for 30minutes.

    8. Centrifuge at 12,000g for 15 minutes.

    9. Carefully pour off the supernatant, wash the pellet with 1 ml of 70% Ethanol.

    10. Air-dry briefly and resuspend in 25 ul of H2O (approximately 0.5 ug/ ul).
  • Determine the exact DNA concentration by absorption spectroscopy at 260 nm.




  • NOTES




    KIT INFORMATION




    REFERENCES



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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