Direct ligation of PCR products for cloning and sequencing
نویسندگان
چکیده
منابع مشابه
T-cassette ligation: a method for direct sequencing and cloning of PCR-amplified DNA fragments.
We describe a method to ligate a PCR-amplified DNA fragment with T-protruding cassettes, which have multiple sites for endonuclease, promoter sequences of T3 and T7, and annealing sites for the universal M13 forward and reverse primers. This method, which we named T-cassette ligation, substantially facilitated direct sequencing and subcloning of PCR products. Two T-cassettes with a protruding T...
متن کاملXcmI-containing vector for direct cloning of PCR products.
Many different strategies are used for cloning polymerase chain reaction (PCR) products. Some use restriction sites pre-integrated into primers or contained in a generated fragment. Others, such as the one used by Stratagene (La Jolla, CA, USA) in its pCR-Script Direct SK(+) Cloning Kit, are based on a blunt-end ligation. These strategies require the use of additional enzymes to polish the end...
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We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplific...
متن کاملDirect cloning of PCR products amplified with Pwo DNA polymerase.
The cloning of polymerase chain reaction (PCR) products provides one with a stable form of the amplified segment and facilitates further manipulation and study of the target molecule. A number of cloning protocols have been developed either based on a restriction endonuclease recognition site built into the primers or by using the template-independent terminal transferase activity (“extendase” ...
متن کاملDirect sequencing of PCR products using unlabeled primers.
An improved protocol is described for using lambda exonuclease to directly sequence PCR products. It is important not to execute PCR cycles beyond the plateau of amplification. The asymmetric PCR and double-stranded DNA sequencing by a snap-cooling procedure were also performed using the same DNA samples and primers. The improved method was the most reliable and produced the best results.
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ژورنال
عنوان ژورنال: Nucleic Acids Research
سال: 1992
ISSN: 0305-1048,1362-4962
DOI: 10.1093/nar/20.23.6427