Wizard® Genomic DNA Purification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For blood DNA isolation, blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Protocol tips
For blood DNA isolation, blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Publication protocol

Cloning, expression, and purification of HP1286
The construct for expression of sequence encoding amino acids 18 to 182 of HP1286 was generated by cloning of the PCR product at NdeI and XhoI sites of the vector pET28b+ (Novagen). The genomic DNA from H. pylori 26695 was used as template in the PCR reaction. Primers used were: 5′-GCGAGCTCATATGAAACCTTATACGATTG-3′ (Forward) and 5′-AAACTCGAGTTGGGCGTAAGCTTCTA-3′ (Reverse). Induction of the protein was carried in E. coli BL21 (DE3) at 37°C for 3–4 h with the addition of isopropyl thio-β-d-galactoside (100 μM) in Luria-Bertani (LB) media. Hexa-His-tagged HP1286 was purified from the soluble fraction by chromatography on Talon resin (Clontech). To remove imidazole, the purified protein was dialyzed overnight followed by another purification step using an endotoxin removal column (Pierce). Amount of endotoxin in the purified protein was assessed by an endotoxin detection kit (Pierce) and the purified protein was concentrated using Amicon Ultra-4 centrifugal filter (Millipore). After staining with the Coomasie brilliant blue dye, the proteins appeared as a single band under reducing conditions. For the generation of an irrelevant-His-tagged (Irr-His) protein, another H. pylori gene (HP1563) was cloned in the same vector pET28b+ at Nde1 and XhoI sites. Irr-His was overexpressed in E. coli BL21 (DE3) and purified from the soluble fraction. The purified protein was of expected size (24 kDa).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA isolation / purification Bacteria - Gram negative Helicobacter pylori using Wizard® Genomic DNA Purification Kit from Promega.

Manufacturer protocol

Download the product protocol from Promega for Wizard® Genomic DNA Purification Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA isolation / purification Bacteria - Gram negative Helicobacter pylori using Wizard® Genomic DNA Purification Kit from Promega. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.