An improved method for cloning PCR fragments.
نویسندگان
چکیده
منابع مشابه
Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments
The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. ...
متن کاملT-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 کاملTurbo cloning: a fast, efficient method for cloning PCR products and other blunt-ended DNA fragments into plasmids.
The method uses a novel plasmid vector, p9lox5, containing a site-specific recombination sequence lox from the lox/Cre recombinase system of bacteriophage P1. There are two distinct stages. Firstly, vector and fragment DNAs are ligated intermolecularly under conditions of macromolecular crowding (15% polyethylene glycol 6000) which accelerate blunt-end joining a thousandfold. Secondly, circular...
متن کاملAn improved PCR method for walking in uncloned genomic DNA.
Several PCR-based methods are available for walking from a known region to an unknown region in cloned or uncloned genomic DNA. The methods are of three types: inverse PCR (1), randomly primed PCR (2) and adaptor ligation PCR (3-6). However, these methods have not been generally applied to walking in uncloned genomic DNA because they are either complicated or inefficient. Recent improvements to...
متن کاملCloning and analysis of PCR-generated DNA fragments.
Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: Genome Research
سال: 1992
ISSN: 1088-9051
DOI: 10.1101/gr.2.1.81