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These are some of the basic techniques used in harvesting clones, preparing stock virus preparations, and preparing infected cultures from M13 and M13 derivatives. The infected cultures can be used for a variety of purposes, such as ssDNA isolation, figure 8 tests, rapid RF isolation, etc.
YT can be substituted with LB, L broth, or B broth. For subsequent preparations, 100ul stock virus can be substituted in place of a picked plaque. For large-scale RF isolations, this technique can be scaled-up to several liters without modification, if care is taken to assure good aeration.


  1. Preparing infected cultures:

    1. Inoculate 20ml YT (in a 125ml flask) with 0.4ml of an overnight culture of E.coli JM103. Incubate at 37C with vigorous agitation for 1-1.5 hr.
    2. Using a sterile pasteur pipette, remove a plug of agar containing a well isolated plaque.
    3. Transfer the plaque to the 20ml culture, and rinse off the inner walls of the pipette tip by drawing the culture back-and-forth through the pipette.
    4. Incubate as before for 5-7 hr. This is then an infected culture, which can be stored for several days at 4C until continuing.
    5. Fill a 1.5ml microfuge tube with infected culture, and spin for 1 min.
    6. Transfer the supernatant to a sterile 1.5ml microfuge tube, and heat for 15 min. at 65C. This is the stock virus preparation, and should have a titer of about 1-2 X 10/12.
    7. Store at 4C.

  2. Rapid ssDNA isolation:

    1. Mix 45ul stock virus with 5ul 2% SDS in a 500ul microfuge tube.
    2. Add 5ul 10X tracking dye and separate on a 1-1.5% agarose gel.

  3. Rapid RF DNA isolation:

    1. RF DNA can be isolated from the pellet in step 6 by any rapid plasmid isolation technique, such as the boiling method described in the CSH Molecular Cloning manual (pages 366-367).


Zinder and Boeke 1982 Gene 19:1-10
Messing,J. 'Recombinant DNA Techniques', Meth. in Enz. (in press)
Messing,J. 'New M13 Vectors for Cloning' , NEN booklet 1982

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