Wizard® Genomic DNA Purification Kit

DNA isolation / purification Bacteria - Gram negative Helicobacter pylori

Experiment
DNA isolation / purification Bacteria - Gram negative Helicobacter pylori
Product
Wizard® Genomic DNA Purification Kit from Promega
Manufacturer
Promega

Protocol tips

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).

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Discussion

Discussion

2 years ago

Author: Israel

How can I improve my DNA yield?

The DNA concentration after using this DNA isolation kit is sometimes too low and thus it is not sufficient for my follow-up experiments. How can I improve it?

Discussion

2 years ago

Author: Milena Alexeyeva Russian Federation

Tips on storing DNA templates?

Hello there! I just started doing experiments on bacterial DNA and I would like your opinion on storing DNA templates. Which are the desired and most optimal conditions?

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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.

Paper title
Secreted Protein HP1286 Triggers Apoptosis in Macrophages via TNF-Independent and ERK MAPK-Dependent Pathways
Full paper
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Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

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