Molecular Techniques and Methods

Screening Lamda Phage Library

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The basic principle of screening recombinant DNA libraries is that bacteriophage plaques contain relatively large amounts of insert DNA that can be detected either directly by hybridization or indirectly by the protein that may be expressed from the cloned segment. The First step in the nucleic acid hybridization screening procedure is to grow large numbers of colonies or plaques on agar plates. Replica copies of these colonies are transferred to nylon membranes, where they can be screened.




MATERIALS AND SOLUTIONS

7% Top Agarose (100 ml)
Agarose --------------------- 7 g
LB -------------------------- 100 ml


Denaturation Solution (100 ml)
0.2 M NaOH --------------- 4 ml of 5 M NaOH
1.5 M NaCl ----------------- 30 ml of 5 M NaCl
Distilled H2O ---------------- 100 ml


Neutralization Solution (100 ml)
0.4 M Tris-HCl (pH 7.6) ---- 40 ml of 1 M Tris-HCl
2 x SSC --------------------- 10 ml of 20 x SSC
Distilled H2O ---------------- 50 ml


Wash Solution (100 ml)
2 x SSC --------------------- 10 ml of 20 x SSC
Distilled H2O ---------------- 50 ml


Hybridization Solution

Total Volume
10 ml
20 ml
20 X SSC
2.4 ml
4.8 ml
1 M Tris-HCl (pH 7.6)
0.2 ml
0.4 ml
100 X Denhardt's Solution
0.1 ml
0.2 ml
Distilled H2O
5.2 ml
10.4 ml
50% Dextran Sulfate
2 ml
4 ml
10% SDS
0.1 ml
0.2 ml

  • Add the SDS last.
  • The dextran sulfate should be of high quality.


    Low-Stringency Wash Buffer (1 liter)
    2 x SSC -------------------------- 100 ml of 20 x SSC
    0.1% SDS ------------------------ 10 ml of 10% SDS
    Distilled H2O ---------------------- 890 ml


    High-Stringency Wash Buffer (1 liter)
    0.2 x SSC ------------------------- 10 ml of 20 x SSC
    0.1% SDS ------------------------- 10 ml of 10% SDS
    Distilled H2O ---------------------- 890 ml


    Autoclaved Salmon Testes DNA (2 mg/ml)
  • Resuspend 1 g salmon testes DNA in 50 ml H2O.
  • Autoclave DNA for 20 min.
  • This process denatures and nicks DNA strands.
  • After autoclave, dilute the DNA to a final concentration of 2 mg/ml.
  • Store at -20oC.




    PROCEDURES

    Plating Bacteriophage Library for Screening

    1. Determine the titer of the phage library by serial dilution.

    2. Mix recombinant phage and plating bacteria in a culture tube and incubate 20 min at 37oC.

    3. Add 7% Top Agarose to the culture tube and transfer mixture to LB plates.
    Disperse bacteria and agarose on plates by tilting the plates back and forth.

    4. Incubate plates at 37oC until plaques cover the plate but are not confluent. Incubation time varies between 6 and 12 hr and depends on type of phage and bacteria used. Store at 4oC.
  • Do not incubate unattended overnight, but rather place at 4oC and allow to continue growth the next day.
  • Allowing phage plaques to incubate for the correct amount of time is critical.

    5. Incubate plates at 4oC for at least 1 hour before applying filters.


    Blotting to Nylon Membranes

    6. Label nylon membranes with a ballpoint pen and apply face down (ink side up) on cold LB plates bearing bacteriophage plaques.
  • This is best accomplished by touching first one edge of the membrane to the agarose and progressively laying down more of the membrane as it wets. Bubbles should be avoided.

    7. Leave membranes on plates for 1-10 min to allow transfer of phage particles to the membrane. During this transfer period the orientation of the membrane to the plate is recorded by stabbing a 20-G needle through the membrane into the agar at several asymmetric points around the edge of the plate. Up to five replicas can be made from each plate.

    8. Remove the membrane slowly from the plate with blunt, flat forceps and place face up on paper towels or filter paper.

    9. Air dry the membrane on the benchtop for 10 min.


    Denaturation and UV-Cross Linking of DNA on Nylon Membranes

    10. Place Whatman 3MM paper on the benchtop and saturate with Denaturation Solution.
    Place membranes on the paper face up for 1-2 min.

    11. Place the second Whatman 3MM paper on the benchtop and saturate with Neutralization Solution.
    Place membranes on the paper face up for 1-2 min.

    12. Place the third Whatman 3MM paper on the benchtop and saturate with Wash Solution.
    Place membranes on the paper face up for 1-2 min.

    13. Air dry the membranes for 1 hour.

    14. UV cross-link the DNA on nylon membranes using UV cross-linker.

    15. Store at room temperature in folded paper towels or other absorbent paper until needed for hybridization.


    Hybridization with Labeled Probes

    16. Wet membranes with Hybridization Solution.
    Lay a membrane bearing plaques on top of 5-20 ml of Hybridization Solution and allow solution to seep through membrane.
    Wet each membrane in turn, producing a stack of wet membranes.
  • When multiple membranes are to be hybridized to the same probe, no more than twenty 8.2-cm discs or ten 20 x 20 cm square membranes should be placed in one stack.

    17. Transfer the stack of wetted membranes to an appropriately sized plastic bag.
    Add enough Hybridization Solution to generously cover membranes and seal.

    18. Prehybridize membranes by placing the bag in a 65oC incubator for at least 1 hour.

    19. While membranes are prehybridizing, pipet the radioactive probe into a 15 ml tube, add 2 mg (1 ml) salmon testes DNA, and boil 10 min.
  • The amount of probe used is important, and should be in the range of 1 to 15 ng/ml of hybridization reaction.

    20. Add the least amounts of Hybridization Solution to the boiled probe.

    21. Remove bag containing membranes from the 65oC incubator.
  • Open bag and remove Hybridization Solution.

    22. Add probe mixture in fresh Hybridization Solution to the bag, exclude as many bubbles as possible, and reseal.

    23. Replace bag in the 65oC incubator and let hybridize overnight.

    24. Remove bag from the 65oC incubator. Cut bag open and squeeze Hybridization Solution out of the bag.

    25. Quickly immerse the membranes in 100-200 ml Low-Stringency Wash Buffer at room temperature in a glass baking dish.
  • Separate all the filters, as they may stick together during hybridization.

    26. Rinse the membranes three times with 100-200 ml Low-Stringency Wash Buffer. Let the membranes sit 10 to 15 min at room temperature in Low-Stringency Wash Buffer with each rinse.

    27. Pour off the Low Stringency Wash Buffer and pour in 100-200 ml High-Stringency Wash Buffer.

    28. Replace the High-Stringency Wash Buffer with another 100-200 ml of High-Stringency Wash Buffer, then place the glass dish containing the membranes in incubator at wash temperature.
  • Usually 15 to 20 min at the desired wash temperature is sufficient to remove most of the background radioactivity.

    29. Remove membranes and mount them either wet or dry on a plastic backing. If the membrane is to be exposed wet, then isolate it from the film by covering it with plastic wrap.

    30. Mark the membranes with radioactive tape to assist in alignment and autoradiograph.
  • X-ray intensifying screens greatly decrease the amount of exposure time required.




    NOTES

  • Recommended Mixtures for Plating Bacteriophage Libraries

    82 mm Plate
    150 mm Plate
    Bacteria
    0.2 ml
    0.5 ml
    Phage
    5,000 pfu
    20,000-30,000 pfu
    Top agarose
    3 ml
    7 ml


  • To prevent recombination between different phage, do not allow them to overgrow, and grow them in recombination-minus hosts where possible. Calculations or the amount of phage stock to be used per plate should be based on a recent titration, and plating cells should be fresh.

  • One important source of background hybridization to membranes is due to the hybridization of the probe to vector sequences or to E. coli DNA. Be certain that there is no vector or E.coli DNA sequences in the probe.

  • Salt concentration. The lower the salt concentration, the higher the stringency.

  • Probe. The nucleic acid probe must be of high specific activity and greater than 50 bp in length so that it can form stable hybrids.




    KIT INFORMATION




    REFERENCES

  • Benton WD, Davis RW (1977) Screening Lamda gt recombinant clones by hybridization to single plaques in situ. Science 196: 180.

  • Denhardt D (1966) A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23: 641-646.

  • Grunstein M, Hogness D (1975) Colony Hybridization: A method for the isolating of cloned DNA's that contain a specific gene. PNAS 72: 3961.

  • Southern EM (1975) Detection of specific sequence among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.

  • Williams BG, Blattner ER (1980) Bacteriophage lamda vectors for DNA cloning. In "Genetic Engineering, Vol. 2 (JK Setlow, A Mullander, eds.) p.201. Plenum, NY.


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

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