The gel-retardation was performed as described previously [26] (link). The LcrF protein was expressed in E. coli BL21(DE3) by pET28a plasmid carrying the lcrF gene from YPIII (named pET28a-LcrF), and purified by Ni-NTA resin. About 400 ng of each DNA fragment, which was amplified from the YPIII genomic DNA, was incubated with various amounts of purified LcrF protein in 20 μl of binding buffer (20 mM Tris-HCl pH 7.4, 4 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, 10% glycerol, and 100 ng bovine serum albumin). A ∼200 bp fragment amplified from the non-coding region upstream of the pYV0053 gene was used as a negative control. Probe binding were performed at 37°C for 1 h, and were then loaded onto a 6.5% native polyacrylamide gel. Electrophoresis was performed in 0.5×TBE buffer on ice. The gel was stained with ethidium bromide for 20 min and scanned using a Syngene GeneGenius gel documentation system.
Genegenius gel documentation system
Manufactured by Syngene
The GeneGenius gel documentation system is a laboratory instrument designed for capturing and analyzing images of DNA, RNA, and protein gels. It provides a reliable and efficient way to document and analyze electrophoresis results.
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Lab products found in correlation
2 protocols using genegenius gel documentation system
1
LcrF Protein Binding Assay
2
Identification of Acinetobacter baumannii by PCR
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From July 2011 to January 2013, a total of 124 non-duplicated A. baumannii isolates were collected from various clinical specimens in two teaching hospitals in Ahvaz, south-est of Iran. Bacterial isolates were initially identified as A. baumannii by biochemical tests (13 ). Suspected isolates were confirmed by PCR to identify blaOXA-51-like gene with specific primers (listed in Table 1 ) to amplify a 353 base pair sequence (14 (link)). DNA template for PCR was obtained by boiling method (15 (link)). Each reaction was carried out in a final volume of 25 µL containing 1x PCR buffer, 1 U Taq polymerase, 1.5 mM MgCl2, 200 µM of dNTP (SinaClon, Iran), 10 pmol of each primer (Eurofins MWG Operon, Germany) and 1 µL of the extracted DNA. PCR conditions were programmed in Mastercycler Eppendorf (Eppendorf, Germany) as follows: Initial denaturation at 94°C for 3 minutes; 35 cycles of 94°C for 45 seconds, annealing 57°C for 45 seconds, extension 72°C for 1 minute and final extension 72°C for 5 minutes. PCR products were separated on 1.5% agarose gel (SinaClon, Iran) by electrophoresis, stained with ethidium bromide (SinaClon, Iran) and then visualized under UV illumination (Syngene GeneGenius gel documentation system). Acinetobacter baumannii ATCC 19606 was used as positive control (14 (link)).
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