Clontech PT3139-1 Manuel de l'utilisateur - Page 10

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CLONTECH Laboratories, Inc.

IV. Advantage-HF PCR Kit continued

4. Good PCR practices

a. Prepare reactions with dedicated pipettors in a dedicated work space

Due to the tremendous amplification power of PCR, minute amounts

of contaminating DNA can produce nonspecific amplification; in

some instances, contaminants can cause DNA bands even in the

absence of added template DNA. We recommend that you set up

your PCR reactions in a dedicated lab area or noncirculating

containment hood and use dedicated pipettors, PCR pipette tips

with hydrophobic filters, and dedicated solutions. Perform post-

PCR analysis in a separate area with a separate set of pipettors.

b. Pipetting

Because of the small volumes used in PCR experiments and the

potential for tube-to-tube variation, careful pipetting technique is

extremely important. Always be sure that no extra solution is on the

outside of the pipette tip before transfer. When adding solution to a

tube, immerse the tip into the reaction mixture, deliver the solution,

and rinse the pipette tip by pipetting up and down several times.

c. Use a Master Mix

Using a Master Mix greatly reduces tube-to-tube variation. There-

fore, use a Master Mix whenever you set up multiple PCR reactions.

If multiple templates are being tested with the same primers, include

the primers in the Master Mix. If one template is being tested with

multiple primer sets, include the template in the Master Mix. For

several sets of parallel samples, assemble multiple master mixes

(e.g., each with a different set of primers). The Master Mix should

be thoroughly mixed before use (i.e., vortexed without bubbling).

d. Include positive and negative controls (i.e., H

template)

5. Touchdown PCR

We have found that "touchdown" PCR significantly improves the

specificity of many PCR reactions in a wide variety of applications

(Section V.B.; Don et al. , 1991; Roux, 1995). Briefly, touchdown PCR

involves using an annealing/extension temperature that is several

degrees (typically 3–10°C) higher than the T

initial PCR cycles (typically 5–10). The annealing/extension tempera-

ture is then reduced to the primer T

6. TaqStart Antibody provides automatic hot start PCR

Do not use a manual hot start or wax-bead-based hot start when using

Advantage-HF. As discussed in the Introduction, hot start is automatic

with Advantage-HF because the enzyme mix already contains TaqStart

Antibody.

page

Protocol # PT3139-1

Version # PR76834

for the remaining PCR cycles.

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

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

O instead of DNA

of the primers during the