Cloning and Labeling of EPSPS Gene

Sequences of A. palmeri EPSPS gene (GenBank accession no. JX564536) were used to develop the PCR primers for cloning of the EPSPS gene. The PCR product was cloned in 2.1-TOPO TA vector (Invitrogen), and the clone was labeled with digoxigenin-11-deoxyuridine triphosphate (Roche Diagnostics) using a standard nick translation reaction. The clone, maize 5S rDNA (54 (link)), was labeled with biotin-16-dUTP (Roche). The BAC clones were labeled with either biotin-16-dUTP or digoxigenin-11-dUTP using a nick translation reaction. Biotin- and digoxigenin-labeled probes were detected with Alexa Fluor 488 streptavidin antibody (Invitrogen) and rhodamine-conjugated antidigoxigenin antibody (Roche), respectively.

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Koo D.H., Molin W.T., Saski C.A., Jiang J., Putta K., Jugulam M., Friebe B, & Gill B.S. (2018). Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri. Proceedings of the National Academy of Sciences of the United States of America, 115(13), 3332-3337.

Publication 2018

digoxigenin-11-deoxyuridine triphosphate biotin-16-dUTP Alexa Fluor 488 streptavidin antibody rhodamine-conjugated antidigoxigenin antibody

Alexa fluor 488 Antibody Biotin Biotin 16 dutp Clones Diagnostics Digoxigenin Digoxigenin 11 dutp Epsps Gene Maize Primers Rdna Rhodamine Streptavidin Topo Vector

Corresponding Organization :

Other organizations : Kansas State University, United States Department of Agriculture, Agricultural Research Service, Clemson University, Michigan State University

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Variable analysis

independent variables

Sequences of A. palmeri EPSPS gene (GenBank accession no. JX564536) used to develop the PCR primers for cloning of the EPSPS gene
Labeling of the clone and maize 5S rDNA (54) with digoxigenin-11-deoxyuridine triphosphate and biotin-16-dUTP, respectively
Labeling of the BAC clones with either biotin-16-dUTP or digoxigenin-11-dUTP

dependent variables

Cloning of the EPSPS gene
Detection of biotin- and digoxigenin-labeled probes with Alexa Fluor 488 streptavidin antibody and rhodamine-conjugated antidigoxigenin antibody, respectively

control variables

Use of 2.1-TOPO TA vector (Invitrogen) for cloning the PCR product
Use of a standard nick translation reaction for labeling the clone and maize 5S rDNA (54)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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