From jwbrown@crab   Wed Jan 15 02:45:08 1992
Subject: PREPARING 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.



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

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



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)