Clontech PT3139-1 사용자 설명서 - 페이지 13
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페이지 인쇄IV. Advantage-HF PCR Kit continued
D. Amplification of Longer Fragments with the Advantage Buffer
The Advantage HF Kit is provided with two buffers—the HF Buffer and the
standard cDNA Buffer. When used with the HF Buffer, this kit delivers the
highest possible fidelity. Fragments of up to ~2.5 kb can be amplified under
these conditions. To amplify longer fragments, some of the increase in
fidelity can be sacrificed to improve elongation efficiency by combining the
HF and cDNA buffers in varying proportions (see Figure 4). To amplify
longer fragments, we recommend replacing the smallest amount of HF
Buffer that allows satisfactory amplification. For example, the 6-kb frag-
ment in Figure 4 can be amplified from a cDNA library in a 50-µl PCR
reaction containing 4 µl of 10X HF Buffer (80% final) and 1 µl of 10X cDNA
Buffer. Optimal conditions for the amplification of other fragments should be
determined individually. We recommend initially trying HF Buffer concen-
trations in the 50–100% range for fragments up to 10 kb.
E. Recommendations for Electrophoresis
We recommend that you transfer a 5-µl sample of your PCR reaction to a
fresh tube and add 1 µl of 5X stop/loading buffer. (The remaining 45 µl of
the reaction mixture can be subjected to further cycling if you do not see a
product.) Analyze your sample(s), along with suitable DNA size markers,
by electrophoresis on a suitable agarose gel containing 0.1–0.5 µg/ml
ethidium bromide. The percentage agarose and the DNA size markers you
choose will depend on the expected range of insert sizes. You may wish to
refer to the following general guidelines before assembling your gel.
Recommendations for agarose gels:
Expected
insert size range
0.3–1.5 kb
0.5–10 kb
>5 kb
TEL:415-424-8222 or 800-662-CLON
FAX:415-424-1064 or 800-424-1350
Recommended
% agarose
1.5
1.2
0.8
Technical Support
CLONTECH Laboratories, Inc.
Recommended
DNA size markers
φX174/ Hae III
1-kb DNA ladder
λ/ Hin d III
Protocol # PT3139-1
Version # PR76834
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