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2. Remove the AmpliTaq DNA polymerase by extracting the aqueous phase twice with 100 µL
of PC9 (see Note 3). Spin for 2 min in a microfuge to separate the lower organic layer
from the upper aqueous layer and transfer the aqueous layer to clean microfuge tube. This
step is essential before digesting the DNA with restriction enzymes for directional cloning
(see Subheading 3.3.) because the polymerase can precipitate, and in the presence of
nucleotides, fill in recessed 3′ termini on DNA.
3. AmAc-EtOH precipitation: To a 100 µL of DNA sample add 50 µL of 7.5 M AmAc (50%
vol). Vortex briefly to mix. Precipitate the DNA with 350 µL of 100% ethanol (2–2.5 vol).
Vortex the samples for 15 s and ice for 15 min. Spin down the DNA at 12,000g for 15 min
at 4°C in a microfuge. Decant the aqueous waste. Add 250 µL of 70% ethanol. Vortex
briefly and spin another 5 min at 4°C. Decant the ethanol and allow the pellets to dry
inverted at room temperature, or dry in a Speed-Vac for 2 to 10 min.
4. Resuspend in 20 µL of PCR water.
5. The next step is to resolve an aliquot (2–10 µL) of the PCR fragments by gel electrophoresis.
Small DNA products (>300 bp) can be resolved at high resolution on 5 to 10% polyacrylamide
gels (12,13). Moderate-sized PCR products (150–1000 bp) should be resolved
on 2 to 4% NuSieve agarose gels (in 1× TAE buffer). Larger PCR products (>500 bp)
can be resolved on 0.8 to 2% agarose gels (1× TAE buffer).
6. After the bromophenol blue dye has reached the end of the gel, soak the gel for 5 to 30 min
in about 10 vol of water containing 1 µg/mL EtBr (see Note 3). Then view and photograph
the gel under ultraviolet light. As shown in Fig. 1, there is little variability in the distance
between the NPA motifs with the known members of the Aquaporin gene family. PCR
amplification of the known Aquaporins cDNAs using the internal degenerate primers
would generate products from 345 to 415 bp. A typical result is shown in Fig. 2.
3.2.3. Secondary PCR Amplifications and DNA Purification
Based on the results from gel electrophoresis of the PCR-amplified DNA products, a
decision must be made on what to do next. The options are the following.
1. Amplify by PCR from the initial DNA sample under different conditions.
2. Amplify by PCR from a different DNA sample under the same conditions. (Different
MgCl 2 concentration, annealing temperature, or primers, see Notes 2, 6, and 7).
3. Gel purify a band(s) of DNA from the gel for cloning or to reamplify by PCR.
4. Purify all PCR-amplified DNA fragments for cloning or to reamplify by PCR.
5. Reamplify by PCR with the same or an internal pair of degenerate primers.
Options 1 and 2 are self explanatory. If you want to gel purify a particular band or
group of bands from an agarose gel, a number of procedures and kits are available (see
Subheading 2.2.). If you plan on immediately cloning a PCR band(s), you may want
to run the rest of the initial PCR on another gel to increase the recovery of DNA. It
is also possible to recover specific DNA fragments from an acrylamide gel (3,12,13).
To purify all PCR-amplified DNA fragments from the remaining sample, a number of
methods are available, including the QIAquick-spin PCR purification kit, which can
be used instead of steps 1–3 in Subheading 3.2.2. (Qiagen). Finally, aliquots of
DNA purified from a gel or from the initial PCR (1–10%) can be reamplified by PCR
with either the same or an internal pair of degenerate oligonucleotide primers (see
Note 1).
When attempting to identify a gene family homolog from a tissue that is known to
express a homolog(s), a number of tricks can be tried to enrich the final PCR sample