15 Cloning Vectors
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10. Applythe supernatant from step 7 or 8 on the QIAGEN-tip and allow it
to enter by gravity flow.
11. Wash the column twice with 2 x 30 ml of buffer QC.
12. Elute the DNA with 15 ml of buffer QF.
13. Precipitate the DNA with 0.7 volumes of isopropanol. Centrifuge the
DNA immediately at 15,000 x g for 30 min at 4°C.
14. Discard the supernatant carefully.
15. Wash the DNA pellet with 15 ml of cold 70% ethanol. Centrifuge at
15,000 x g for 30 min at 4°C.
16. Air dry the pellet for 5 min and dissolve the DNA in a suitable volume of
TE buffer. The maximum binding capacityfor plasmid DNA of the QIAGEN-tip 500 is 500 J.tg.
17. Determine the DNA concentration with the spectrophotometer at an
OD26o·
Subprotocol 6
Large-Scale Preparation of Bacteriophage DNA (Replicative Form (RF))
This method describes the preparation of double-stranded DNA from the
replicative form (RF) ofbacteriophage M13. The procedure is also suitable
for other filamentous bacteriophage such as fd or f1• Furthermore, the preparation method for closed circular DNA described in steps 7-27 can be
used for a large scale plasmid preparation to yield plasmids of high purity.
However, you can also use the column Chromatographie method (Subprotocol 5) to isolate M13-RF-DNA.
Materials
• A single colony of E. coli JM103 grown on a minimal agar plate
• An M13 phage plaque (see Subprotocol 3, Materials)
• CsCl
• Lysozyme
• LB medium (see Subprotocol1, Materials)
Reagents
Salutions