Cloning and analysis of PCR-generated DNA fragments.

Costa, Gina L.; Grafsky, A; Weiner, Michael P.

doi:10.1101/gr.3.6.338

Cited by 24 publications

(10 citation statements)

References 12 publications

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“…[8][9][10][11][12] and harvested by 15 min of centrifugation at 3000g. Pellets were frozen at 2208C overnight, thawed, then resuspended with 600 lL of Buffer A (50 mM Tris-HCl, 50 mM sucrose and 1 mM EDTA, pH 7.5) containing 500 units of Ready-lyse (Epicentre).…”

Section: Microscreening-expression/purificationmentioning

confidence: 99%

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

et al. 2007

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Successful protein expression, purification, and crystallization for challenging targets typically requires evaluation of a multitude of expression constructs. Often many iterations of truncations and point mutations are required to identify a suitable derivative for recombinant expression. Making and characterizing these variants is a significant barrier to success. We have developed a rapid and efficient cloning process and combined it with a protein microscreening approach to characterize protein suitability for structural studies. The Polymerase Incomplete Primer Extension (PIPE) cloning method was used to rapidly clone 448 protein targets and then to generate 2143 truncations from 96 targets with minimal effort. Proteins were expressed, purified, and characterized via a microscreening protocol, which incorporates protein quantification, liquid chromatography mass spectrometry and analytical size exclusion chromatography (AnSEC) to evaluate suitability of the protein products for X‐ray crystallography. The results suggest that selecting expression constructs for crystal trials based primarily on expression solubility is insufficient. Instead, AnSEC scoring as a measure of protein polydispersity was found to be predictive of ultimate structure determination success and essential for identifying appropriate boundaries for truncation series. Overall structure determination success was increased by at least 38% by applying this combined PIPE cloning and microscreening approach to recalcitrant targets. Proteins 2008. © 2007 Wiley‐Liss, Inc.

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Section: Microscreening-expression/purificationmentioning

confidence: 99%

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

et al. 2007

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“…5) Direct cloning of reamplified LDD-PCR-derived cDNAs by T/A ligation into the pGEM-T vector resulted in variable and reduced transformation and recombinant frequency. Low efficiency of T/A cloning suggests either that rTth DNA polymerase has a low 3Ј-terminal deoxynucleotidyl transferase (TdT) activity or that under LDD-PCR conditions the addition of adenosines to the 3Ј terminus of the PCR products may be less frequent (15,16). To increase cloning efficiency of LDD-PCR clones we used the template-independent TdT activity of Taq polymerase, which results in the addition of a single nucleotide to the 3Ј end of most PCR products (16 -18).…”

Section: Comparison Of Cold-and Hot-start Dd-pcr Withmentioning

confidence: 99%

Identification and Cloning of Differentially Expressed Genes by Long-Distance Differential Display

Jurecic

Nachtman

Colicos

et al. 1998

Analytical Biochemistry

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“…Numerous gene cloning methods, including sticky-end cloning (Scharf et al, 1986), blunt-end cloning (Costa et al, 1994), TA cloning (Holton and Graham, 1991;Marchuk et al, 1991), ligation independent cloning (LIC) (Aslanidis and de Jong, 1990;Shuldiner et al, 1990;Hsiao, 1993;Yang et al, 1993;Kaluz and Flint, 1994;Tillett and Neilan, 1999), and site-specific recombination systems (Hartley et al, 2000), have been developed over the years. These cloning approaches are widely used and have proven to be highly efficacious in most instances; however, they present several limitations because of the extensive enzymatic treatment of the required PCR products or vectors.…”

Section: Introductionmentioning

confidence: 99%

Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products

et al. 2015

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ABSTRACT. In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactions commonly included in (2015) routine cloning procedures. A homologous arm is linked to the 5'-end of the forward primer used to amplify both the target gene and vector. A different homologous arm is linked to the 5'-end of the reverse primer. Therefore, genes can be cloned into the vectors by homologous recombination after co-transformation of the amplified target gene and the linearized vector, which bear the same homologous arm on either end. More than twentyfour different plasmids were generated by this method, which uses two simple polymerase chain reaction steps. This method is highly efficient in cloning any gene of interest into any vector at any site without sequence constraints, as no restriction and ligation reactions are required.

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Cloning and analysis of PCR-generated DNA fragments.

Cited by 24 publications

References 12 publications

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

Identification and Cloning of Differentially Expressed Genes by Long-Distance Differential Display

Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products

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