DNeasy Blood & Tissue Kit

Protocol tips

Upstream tips	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

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

solation and Identification of Lactic Acid Bacteria from Fermented Milk
Sampling was done from raw cow milk randomly collected from two different restaurants (Al-Berkat Curry House and Grand City) located in Petaling Jaya, Selangor, Malaysia. The raw milk samples were pasteurized at 63°C for 30 minutes to kill the harmful bacteria present in the raw milk. The milk was left to ferment in a sterile flask under room temperature for two days. After fermentation process, LAB was isolated by growing on de-Mann, Rogosa and Sharpe (MRS) agar plates (Merck, Germany). These plates were incubated for 24 hours at 37°C. Single colonies were picked from the plates and sub-cultured two times to get a pure colony based on morphology. The isolates were grown in MRS broth for 24 hours and centrifuged at 10,000 x g for 20 minutes. The supernatant from each isolate was used for preliminary antimicrobial test by using agar well diffusion assay. 50 μl of the supernatant from each isolate was transferred to different wells on a Mueller-Hinton agar plate seeded with indicator bacteria. The bacteria isolate (A3) that produces potent antimicrobial activity was selected for further tests. For identification of bacteria, five tests were done including Gram-stain, spore stain, catalase test, oxidase test and the ability to grow on bile esculin azide agar (Merck, Germany). Cell morphology and Gram-stain were examined by light microscope at 1000X magnification. Schaeffer and Fulton’s spore staining method modified by [] was followed. Bile esculin azide agar tested the ability of the bacteria to hydrolyze esculin in the presence of bile. Bacteria positive for esculin hydrolysis can hydrolyze the glycoside esculin to esculetin and dextrose. The bile in the agar was used to inhibit Gram-positive bacteria other than enterococci while the sodium azide inhibited Gram-negative bacteria. LAB was identified using API 50CHL (bioMérieux, France) which tested the ability of the bacteria to metabolize 49 kinds of carbohydrates. Pure colonies of the bacteria were transferred into the test medium and loaded into the test strip. The test strip was incubated for 48 hours at 37°C. The result obtained was examined for bacterial similarity to the database using the API software provided. The bacteriocin producer strain was identified by 16S rRNA gene sequencing using universal primers namely 27F () and 1492R (). PCR reaction mixture (25 μl total) contains 2.5 μl PCR buffer, 2 μl of dNTP mix (2.5mM each), 1μl of each primer (20 pmol), 100ng of DNA template, 0.5 μl of i-Taq™ DNA polymerase 5U/μl supplied from iNtRON Biotechnology, Korea. PCR conditions used initial denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 52°C for 1 minute, extension at 72°C for 1.5 minutes for 30 cycles with a final extension at 72°C for 10 minutes.

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