An improved thermal cycle for two-step PCR-based targeted mutagenesis
نویسندگان
چکیده
منابع مشابه
An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes.
The QuikChangeTM protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by u...
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The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully ca...
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with impervious exteriors, such as shells or chitinous exoskeletons. Advantages to this method are high throughput, high success rate and enough template DNA for several different assays (FRE-PCR used only 5 μL of the 20-μL supernatant). Other extraction methods that were tried and discarded for low success rates included boiling in water or PCR buffer, grinding before boiling, grinding with co...
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ژورنال
عنوان ژورنال: Nucleic Acids Research
سال: 1992
ISSN: 0305-1048,1362-4962
DOI: 10.1093/nar/20.18.4934