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●●●●●　Spot blots　●●●●●



A rapid method for screening a large number of DNA samples for homology to a known DNA is to hybridize the known probe DNA (radioactively labeled) to filters to which the test DNAs how been spotted in a grid pattern. The samples that hybridize to the probe will form a spot upon autoradiography. For example, the spot test can be used to screen a series of hybrid M13 or plasmid isolates for the presence of the desired sequences. Because it is used as a method for screening many samples at once, the spotted DNA samples are usually rapid plasmid isolates, rapid ssDNA isolates from M13, or other crude isolates. It is noteworthy that this method can be used either with ds or ssDNA, or both at the same time.

  1. Materials

    1. Denaturing solution (per liter)
      final conc.
      4.00 grams NaOH 0.1M
      87.67 grams NaCl 1.5M

    2. Neutralizing solution (per liter)
      final conc.
      60.57 grams Tris-OH, pH 7 0.5M
      175.3 grams NaCl 3M

  2. Methods

    1. Mark off a grid on a piece of 3MM filter paper with the lines 1-2cm apart, with enough intersections for all of the test samples and controls. Number each intersection, and make a list relating the intersection numbers to specific test samples.
    2. Cut a peice of nitrocellulose big enough to cover the grid with a 2 cm margin, and make an orienting mark on the sheet.
    3. Lay the marked-off 3MM paper on a light box, and over this place the nitrocellulose filter. With the light on, the grid should be clearly visible through the filter.
    4. Spot 2-10ul (5ul is good) of each sample onto the filter at the appropriate positions. Allow the filter to air dry.
    5. Float the filter on denaturing solution for 30 min. at room temperature.
    6. Carefully transfer (float) the filter to neutralizing solution. Incubate for 30 min. at room temperature.
    7. Transfer the filter onto a piece of 3MM paper, and allow to air dry.
    8. Incubate the filter for at least 1 hr at 80C in a vacuum oven (if the filter is heated in air, it may burn). The filter is now ready for hybridization. Store at room temperature in a dark, cool place.


Maniatis, et al. 'Molecular Cloning', CSH 1982
Messing 'New M13 Vectors for Cloning" NENbooklet
Messing 'Recombinant DNA Techniques' Meth. in Enz (in press)

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