Clontech PT3139-1 Manuel de l'utilisateur - Page 13

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IV. 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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