Figure 8 (C) test

●●●●●　Figure 8 (C) test　●●●●●

The figure-8 test is a test that identifies M13 clones that contain complementary sequences in their viral ssDNA. This occurs when the two clones contain the same region of DNA inserted in opposite orientations. This can be used to identify clones containing the same DNA fragment inserted in opposite directions, to identify clones containing nonidentical but overlapping complementary regions, or to find clones containing portions of DNA also found (complementary) in a larger clone.
A good positive control for this test is M13mp8 against M13mp9, since they contain a 33 bp region inserted in opposite orientation (the polylinker). If properly done, this mixture will show a faint figure-8 band upon electrophoresis.

NOTE: Broken circles are usually faintly visible in front of the circular ssDNA, and do not interfere with the test.
NOTE: The viral DNA does not interfere because it is all (+) strand.

Methods

Prepare ssDNA from each strain to be tested by mixing 0.4ml stock virus (1-5 X 10/12 pfu/ml) with 50ul 2%SDS and 50ul 10X tracking dye. Vortex.

Mix the strains to be tested against each other (do this in duplicate) by adding 20ul of each of the two ssDNAs in a small microfuge tube and vortex. Add 100ul of sterile mineral oil to each tube, and centrifuge 5 sec.

Store one tube of each duplicate set at 4C. Incubate the other in a 68C water bath for 1 hr to allow the annealing of any complementary regions.

Run the duplicate samples side-by-side on a 1-1.5% agarose gel. Also run a lane containing M13mp8 ssDNA, as a standard. Those lanes containing a band with reduced mobility in the heated samples, but not in the unheated samples, are those with figure-8s'and therefore contain complementary sequences.

Messing,J. 1983 'Recombinant DNA Techniques' Meth. in Enz. (in press)
Messing,J. 1982 'New M13 Vectors for Cloning' NEN Booklet