Recovery of Unlabeled PCR Product from Polyacrylamide Gel for Sequencing
نویسندگان
چکیده
منابع مشابه
Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.
Numerous methods have been developed for direct sequencing of single or double-stranded DNA amplified by PCR, and most require template purification steps prior to sequencing to remove excess unincorporated primers and dNTPs. Purification methods can be cumbersome, laborious and time consuming, or yield low DNA recoveries. Strategies for direct sequencing of doublestranded DNA products without ...
متن کاملElectrophoresis of unlabeled proteins in a sequencing gel apparatus.
In an earlier issue of this journal, Whelan and Taylor (4) described the adaptation of the Sequi-Gen electrophoretic cell (Bio-Rad Laboratories, Hercules, CA, USA) for analysis of proteins. Their paper demonstrated that, with slight modifications of the plate assembly and gel loading protocols, it was possible to obtain a large degree of separation of the radiolabeled polypeptides electrophore...
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.
متن کاملPolyacrylamide Gel Electrophoresis
Polyacrylamide gels are typically formed by polymerization of the monomer acrylamide crosslinked to the co-monomer, N,N’-methylenebis-acrylamide, commonly called BIS. This process is a freeradical polymerization that requires an initiator, usually ammonium persulfate, and a catalyst like N,N,N’,N’-tetraethylmethylenediamine (TEMED). A distinct advantage of polyacrylamide gel systems is that by ...
متن کاملNeutral polyacrylamide gel electrophoresis.
MATERIALS 5x TBE electrophoresis buffer Polyacrylamide gels are poured and run in 0.5x or 1x TBE at low voltage (1-8 V/cm) to prevent denaturation of small fragments of DNA by Joulic heating. Other electrophoresis buffers such as 1x TAE (please see Agarose Gel Electrophoresis) can be used, but they are not as good as TBE. The gel must be run more slowly in 1x TAE, which does not provide as much...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 1996
ISSN: 0736-6205,1940-9818
DOI: 10.2144/96213bm02