Electrophoresis - Orienting M13 Bacteriophage Inserts

Jeff Lawrence

1. Amplify M13 phage in 2XYT.

2. Centrifuge 1.5 mL cells for 3 min at 4°.

3. Mix:
	10 µL	Supernatant from lysate 1
	10 µL	Supernatant from lysate 2
	5 µL	6X Loading Dye
	1 µL	20% SDS
	3 µL	1 M NaCL
4. Heat to 65° for 10 min.

5. Cool slowly to 22° over 30 min

6. Load samples on an agarose gel and separate DNA by electrophoresis.

7. Phage with oppositely oriented inserts will form an addition semi-duplex DNA band due to hybridization of the oppositely oriented inserts.

8. To insure isolating clones of both orientations, isolate 8 clones and test each with the other seven as well as itself.

Last Update: Thursday, 19-Jun-2014 11:50:30 PDT
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Eric Kofoid eckofoid at ucdavis.edu