Clontech PT3139-1 Gebruikershandleiding - Pagina 9

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IV. Advantage-HF PCR Kit Protocol

PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.

A. General Considerations

1. Thermal cycler

The guidelines for cycling parameters in this protocol have been

developed for a Perkin-Elmer DNA Thermal Cycler 480 or 9600 and

Perkin-Elmer GeneAmp 0.5-ml PCR reaction tubes. Newer cyclers

may allow for shorter cycle times and eliminate the need to use oil.

The optimal cycling parameters may vary with different templates,

primers, experimental protocols, tubes, and thermal cyclers.

Refer to the Troubleshooting Guide (Section V) for suggestions on

optimizing PCR conditions.

2. Primer design

Primer design is the single largest variable in PCR applications and the

single most important factor in determining the success or failure of

PCR reactions. Always check and recheck your primer design before

constructing or ordering primers. CLONTECH offers PRIMER

PREMIER (#V1072-1, V1079-1), powerful, easy-to-use software that

ensures optimal primer design.

Length and G/C content: The Advantage-HF PCR Kit can be used in

a wide variety of PCR applications, and the constraints on primer

design will vary from one application to the next. In general, however,

primers should have a T

a two-step cycling program with a 68°C annealing/extension step.

Therefore, whenever possible, primers should be at least 22 nucleotides

(nt) long (25–30-mers are preferred) and have a GC content of 45–60%.

3. Template quality

Because of the exponential nature of PCR amplification, many conven-

tional PCR applications work well with templates of average or even

low quality. However, the longer the target, the more important tem-

plate quality becomes. This is because the number of unnicked, full-

length targets decreases as the target length increases, so poor quality

DNA will have very few large, unnicked targets. Furthermore, some

depurination occurs when DNA is denatured during thermal cycling,

and this can lead to strand scission. Therefore, it is particularly

important to prepare high-quality, high molecular weight DNA when

amplifying large targets.

Template quality is also important when the highest possible sensitivity

is needed. Furthermore, in cDNA applications such as RACE and other

RT-PCR protocols, incomplete reverse transcription can lead to an

absence of product, shorter than full-length products, or smearing.

For 5' and 3' RACE and general PCR from cDNA, you can ensure the

quality of your cDNA by using Marathon-Ready cDNA from CLONTECH.

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

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

of at least 70°C to achieve optimal results in

Technical Support

CLONTECH Laboratories, Inc.

Protocol # PT3139-1

Version # PR76834

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