Easy-DNA™ gDNA Purification Kit

DNA isolation / purification Bacteria - Gram negative Pseudomonas aeruginosa

Experiment
DNA isolation / purification Bacteria - Gram negative Pseudomonas aeruginosa
Product
Easy-DNA™ gDNA Purification Kit from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Downstream tips
- Include RNAse treatment for 15-20 min.
- Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C
- Use prewarmed TE buffer to elute the DNA

Publication protocol

. Evaluation of DNA extraction methods
Six type strains were used for the evaluation of DNA extraction methods (Table 1). Among these, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were used as positive control strains; Streptococcus pyogenes was used as an example of a strain that is difficult to lyse, and Haemophilus influenzae was used as a representative of fragile bacteria. Candida albicans was used as negative control. All strains were grown on blood and chocolate agar plates for 24 hr and diluted until appropriate concentrations were reached at a McFarland standard of 0.5. Using distilled water, we performed 10-fold serial dilutions from 108 colony-forming units (CFU)/mL to 100 CFU/mL. All dilutions were centrifuged, and the supernatant was harvested, except when the QIAmp DNA Mini kit (Qiagen GmbH, Hilden, Germany) and the heating method were used, for which we left 200 µL of supernatant on top of the pellets. The commercial DNA extraction kits used in this study were the InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA, USA), Exgene™ Clinic SV kit (GeneAll Biotechnology Co. Ltd., Seoul, Korea), QIAmp DNA Mini kit (Qiagen GmbH), and Easy-DNA™ kit (Invitrogen, Carlsbad, CA, USA). The InstaGene Matrix is composed of 6% InstaGene Matrix and a magnetic stir bar. Boiling in the presence of the matrix resulted in cell lysis. The Exgene™ Clinic SV kit and QIAmp DNA Mini kit utilize the silica-binding technology to purify DNA. The DNA in the lysates binds to the silica membrane, and impurities pass through the membrane into a collection tube. The Easy-DNA kit uses ethanol precipitation purification with chloroform. All procedures were performed according to the manufacturer's instructions. For the heating method, all diluted samples were heated at 100℃ for 10 min and placed on ice for 5 min. After centrifugation, supernatant was used as the source of DNA for PCR analysis. We used the 27F and 515R primer set to compare the DNA extraction methods (Table 2).

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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 Pseudomonas aeruginosa using Easy-DNA™ gDNA Purification Kit from Thermo Fisher Scientific.

Paper title
Evaluation of DNA Extraction Methods and Their Clinical Application for Direct Detection of Causative Bacteria in Continuous Ambulatory Peritoneal Dialysis Culture Fluids from Patients with Peritonitis by Using Broad-Range PCR
Full paper
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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Easy-DNA™ gDNA Purification Kit below.

Download PDF Download manufacturer protocol

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