Clontech PT3139-1 Benutzerhandbuch - Seite 12

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CLONTECH Laboratories, Inc.
IV. Advantage-HF PCR Kit continued
C. Recommended Cycling Parameters
Use the following guidelines when setting up your initial experiments with
the Advantage-HF Polymerase Mix. These are general guidelines—the
optimal parameters may vary with different thermal cyclers and will depend
on your particular primers and templates, and on other experimental
variables. Note: When using the Advantage-HF Kit with products such as
CLONTECH's Marathon
cDNAs, or the Delta
mended in the protocol for that kit.
Cycle Parameters
(PE 480)
• 94°C for 1 min
• 25–35 cycles
• 68°C for 3 min
• Soak at 15°C
a
25 cycles for multiple-copy genes or medium-to-high abundance cDNAs; 30–35
cycles for single- or low-copy-number genes or rare cDNAs. For most applications,
we prefer two-step cycles (denaturation at T
T
) over three-step cycles (denaturation at T
2
extension at T
less than 60–65°C and in certain special protocols (such as Delta Differential Display).
b
Use the shortest possible denaturation time. Exposure of DNA to high temperatures
causes some depurination of single-stranded DNA during denaturation, which
eventually leads to strand scission. High temperature also leads to gradual loss of
enzyme activity.
c
Use the highest possible annealing/extension temperature. See Note a. Shorter
targets may be amplified using shorter extension times.
Some researchers prefer to use an annealing/extension time equal to the expected
target size plus two minutes.
d
Optional: This final extension may reduce background in some cases.
page
Protocol # PT3139-1
12
Version # PR76834
TM
cDNA Amplification Kit, Marathon-Ready
TM
Differential Display, use the parameters recom-
a
b
94°C 30 sec
c
68°C
4 min
d
). Three-step cycles will be necessary when the T
3
Technical Support TEL:415-424-8222 or 800-662-CLON
(PE 9600)
• 94°C for 15 sec
• 25–35 cycles
94°C 5–15 sec
68°C
• 68°C for 3 min
• Soak at 15°C
followed by annealing and extension at
1
followed by annealing at T
1
FAX: 415-424-1064 or 800-424-1350
TM
a
b
c
4 min
d
followed by
2
of the primers is
m