Clontech PT3139-1 Посібник користувача - Сторінка 9
Переглянути онлайн або завантажити pdf Посібник користувача для Лабораторне обладнання Clontech PT3139-1. Clontech PT3139-1 20 сторінок. Advantage-hf pcr kit
Друкувати сторінкуIV. 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
m
Technical Support
CLONTECH Laboratories, Inc.
Protocol # PT3139-1
Version # PR76834
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