Clontech PT3139-1 Manual del usuario - Página 11
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Imprimir páginaIV. Advantage-HF PCR Kit continued
7. Use of additives
TaqStart Antibody binds KlenTaq-1 DNA polymerase with high affinity
under the conditions described in this protocol. The addition of 2–5%
DMSO will not interfere with TaqStart function and may improve results
in some instances (see Section V.A.). However, the addition of
formamide or other cosolvents may disrupt TaqStart function. Further-
more, excessive glycerol, solutes (e.g., salts), pH extremes, or other
deviations from the recommended reaction conditions may reduce the
effectiveness of the antibody and/or DNA polymerases.
B. Control PCR Reactions
The following PCR reactions can be performed in parallel with your
experiments as controls to ensure that the Advantage-HF Kit is working
properly. A positive control template and primers are provided in the kit.
1. Place all components on ice and allow to thaw completely. Mix each
component thoroughly before use.
2. Combine the following reagents in a 0.5-ml PCR tube.
Positive
Control
32 µl
50
3. Mix well and spin the tube briefly to collect all the liquid in the bottom
of the tube.
4. Add 1–2 drops of mineral oil to prevent evaporation during cycling and
cap firmly. A good "capping" of mineral oil should have a well-defined
meniscus between the two phases.
5. Commence thermal cycling. If using a Perkin-Elmer DNA Thermal
Cycler Model 480 or 9600, use the parameters described in Section C
below. 20–22 cycles with a 4-min annealing/extension time is sufficient
for amplification of the positive control template provided in the kit.
6. Transfer a 5-µl sample of your PCR reaction to a fresh tube and add
1 µl of 5X stop/loading buffer. Analyze your sample(s), along with
suitable DNA size markers, by electrophoresis on a 0.8–1.2 % agarose/
ethidium bromide gel.
Expected results: If you are using the positive control reagents provided
in the kit, the reaction should produce a single major band of 2 kb. No bands
should be generated in the negative (i.e., no DNA template) control.
TEL:415-424-8222 or 800-662-CLON
FAX:415-424-1064 or 800-424-1350
Negative
Control
37 µl
5 µl
5 µl
5 µl
---
2 µl
2 µl
5 µl
5 µl
1 µl
1 µl
µ
µ
l
50
l
Technical Support
Purified H
O
2
10X HF PCR reaction buffer
Control DNA template (~0.2 ng/µl)
Control primer mix (10 µM ea.)
10X HF dNTP mix
50X Advantage-HF Polymerase Mix
Total
CLONTECH Laboratories, Inc.
Protocol # PT3139-1
Version # PR76834
page
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