Direct PCR of Intact Bacteria (Colony PCR)

Woodman, Michael E.; Savage, Christina R.; Arnold, William K.; Stevenson, Brian

doi:10.1002/cpmc.14

Cited by 61 publications

(46 citation statements)

References 4 publications

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“…Ligation and transformation of competent cells of Escherichia coli JM109 was carried out according to the manufacturer’s protocol and as described in Sambrook and Russell [28]. To screen for clones with correct inserts, colony PCR was carried out as detailed in Woodman [29] using primers M13-F and M13-R (S1 Table). The PCR program used was 95°C for 3 min, 30 cycles at 94°C for 40 sec, 52°C for 40 sec and 72°C for 1 min, and a final elongation at 72°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model

et al. 2017

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Previously, Planted Fixed-Bed Reactors (PFRs) have been used to investigate microbial toluene removal in the rhizosphere of constructed wetlands. Aerobic toluene degradation was predominant in these model systems although bulk redox conditions were hypoxic to anoxic. However, culture-independent approaches indicated also that microbes capable of anaerobic toluene degradation were abundant. Therefore, we aimed at isolating anaerobic-toluene degraders from one of these PFRs. From the obtained colonies which consisted of spirilli-shaped bacteria, a strain designated 15–1 was selected for further investigations. Analysis of its 16S rRNA gene revealed greatest similarity (99%) with toluene-degrading Magnetospirillum sp. TS-6. Isolate 15–1 grew with up to 0.5 mM of toluene under nitrate-reducing conditions. Cells reacted to higher concentrations of toluene by an increase in the degree of saturation of their membrane fatty acids. Strain 15–1 contained key genes for the anaerobic degradation of toluene via benzylsuccinate and subsequently the benzoyl-CoA pathway, namely bssA, encoding for the alpha subunit of benzylsuccinate synthase, bcrC for subunit C of benzoyl-CoA reductase and bamA for 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase. Finally, most members of a clone library of bssA generated from the PFR had highest similarity to bssA from strain 15–1. Our study provides insights about the physiological capacities of a strain of Magnetospirillum isolated from a planted system where active rhizoremediation of toluene is taking place.

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Section: Methodsmentioning

confidence: 99%

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model

et al. 2017

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“…Pick and plate several individual colonies onto a fresh LB agar plate (see Step 3.1), grow at 28 °C for 48 h, and take a small amount of bacteria for analysis by colony PCR 33 to confirm the presence of the specific binary constructs.…”

Section: Protocolmentioning

confidence: 99%

Identification of Plasmodesmal Localization Sequences in Proteins <em>In Planta</em>

Cheng

Lazarowitz

Citovsky

2017

JoVE

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Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

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“…When selections are complete, clone and sequence the PCR library Form H. Colony PCR (Woodman, 2008) is recommended when selecting colonies for sequencing. Blue-white colony screening can give a false negative result on an insert of this size (56 bp).…”

Section: Strategic Planningmentioning

confidence: 99%

SELMA: Selection with Modified Aptamers

Temme

Krauss

2015

CP Chemical Biology

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In vitro selection of nucleic acid aptamers, coined SELEX, has led to the discovery of novel therapeutics and aided in the structural and mechanistic understanding of many ligand‐biomolecule interactions. A related method, selection with modified aptamers (SELMA), enables selection of DNA aptamers containing bases with a large modification that cannot undergo PCR. A key application of this method is the evolution of aptamers containing carbohydrate modifications. Carbohydrate‐binding proteins normally require several copies of the carbohydrate moiety for strong recognition. Whereas it may be difficult to rationally design synthetic scaffolds that cluster glycans in the optimal spacing and orientation for target recognition, SELMA furnishes glycoaptamers with highly optimized glycan clustering, achieving low‐nanomolar recognition. Although numerous applications can be envisioned, the protocols and discussions in this article describe procedures involved in applying SELMA to the discovery glycoDNAs that bind to the HIV broadly neutralizing antibody 2G12. © 2015 by John Wiley & Sons, Inc.

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Direct PCR of Intact Bacteria (Colony PCR)

Abstract: This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using the polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. © 2016 by John Wiley & Sons, Inc.

Cited by 61 publications

References 4 publications

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model

Identification of Plasmodesmal Localization Sequences in Proteins <em>In Planta</em>

SELMA: Selection with Modified Aptamers

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