Clontech PT3139-1 Podręcznik użytkownika - Strona 5

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I. Introduction continued

The Advantage-HF Kit ensures

high-fidelity amplification of ge-

nomic as well as cDNA tem-

plates. In Figure 3, the Advan-

tage HF Kit was used to amplify

a 2.1-kb fragment of the bovine

pancreas trypsin inhibitor gene

from different amounts of total

calf thymus DNA. The fragment

was efficiently amplified from as

little as 25 ng of genomic DNA

(Lane 4).

Increase elongation efficiency

for longer fragments

Two reaction buffers are included

in the Advantage-HF Kit: the HF

Buffer and the original cDNA

Buffer. Use of the HF buffer de-

livers the highest possible fidel-

ity, as represented in Figure 1.

Fragments of up to ~2.5 kb can

be amplified under these condi-

tions. To amplify longer frag-

ments, some of the increase in

fidelity can be sacrificed to im-

prove elongation efficiency by

combining the HF and cDNA

buffers in varying proportions.

Figure 4, Panel A shows the

fidelity resulting from the use of

varying percentages of the HF

Buffer (see p. 3 for description of

assay). Panel B demonstrates

amplification of a 6.0-kb cDNA

fragment using the indicated con-

centrations of HF Buffer. In this

example, the fragment is suc-

cessfully amplified in 80% HF

Buffer, conditions allowing a ~3-

fold increase in fidelity over the

cDNA buffer. Optimal conditions

for the amplification of other frag-

ments should be determined in-

dividually.

TEL:415-424-8222 or 800-662-CLON

FAX:415-424-1064 or 800-424-1350

ng:

Figure 3. Advantage-HF amplification from genomic

DNA. The indicated amounts of calf thymus DNA were

used to amplify a 2.1-kb fragment of the bovine pan-

creas trypsin inhibitor gene. M: λ/ Hin d III DNA size

markers. Cycling parameters are the same as in Fig-

ure 2.

HF Buffer %:

4.4-

2.0-

Figure 4. The effect of HF Buffer concentration on PCR

fidelity (A) and amplification (B) of a 6.0-kb cDNA

fragment from Human Placenta cDNA. Size markers

are λ/ Hin d III DNA.

Technical Support

CLONTECH Laboratories, Inc.

100

Protocol # PT3139-1

Version # PR76834

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