240
HEINZ-ULRICH G. WEIER
8. Prepare 1.5 ml microcentrifuge tubes (4 tubes per clone with ID on
top).
9. Decant supernatant and air dry the pellet. Resuspend pellet in 3 ml of
1xTE, pH 7.5.
10. Transfer the DNA solution to four 1.5 ml microcentrifuge tubes.
11. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1,
pH 8.0), vortex well and spin at high speed (10,000 g) for 3 min.
12. Transfer the top layer to new 1.5 ml microcentrifuge tubes and add an
equal volume of chloroformlisoamyl alcohol (24:1). Vortex well and
centrifuge at high speed (10,000 g) for 3 min.
13. Transfer the top layer to new 1.5 ml microcentrifuge tubes. Add 40 III of
RNase (1 mg/ml, DNAse free) to each of the four tubes and incubate at
37°C for 30 min.
14. Add 1 volume of isopropanol and gently mix by inversion. Centrifuge
at high speed (10,000 g) for 20 min.
15. Decant supernatant and wash pellet with 1 volume of cold 70% ethanol,
and centrifuge at high speed (10,000 g) for 3 min.
16. Decant the 70% ethanol and air dry the pellet.
17. Resuspend pellet in 30 1111 xTE, and measure DNA concentration after
the pellet is completely dissolved.
Subprotocol 5
Generation of probes by in vitro DNA amplification
In vitro DNA amplification using the polymerase chain reaction (PCR) is a
very efficient method to synthesize probe DNA. It can be applied to amplify a particular DNA sequence, such as a part of the cloning vector, or
with mixed-base primers to perform arbitrary amplification of virtually
any sequence of interest. As illustrated in the following paragraphs, the
former amplification can be applied to prepare DNA landmark probes,
while the latter allows the preparation of probes to counterstain the fibers.