Electrophoresis - Orienting M13 Bacteriophage Inserts
1. Amplify M13 phage in 2XYT.
2. Centrifuge 1.5 mL cells for 3 min at 4°.
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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