DNeasy UltraClean 96 Microbial Kit (384)

DNA isolation / purification Bacteria - Gram negative E.coli

Experiment
DNA isolation / purification Bacteria - Gram negative E.coli
Product
DNeasy UltraClean 96 Microbial Kit (384) from Qiagen
Manufacturer
Qiagen

Protocol tips

Publication protocol

To isolate in vivo-adapted EcN isolates, intestinal contents or fecal samples were vortexed in phosphate-buffered saline and streaked on LB plates containing kanamycin. Single colonies were placed in liquid media and grown at 37 C overnight. Un-adapted EcN strains were also grown overnight. Total DNA was extracted from each culture using the DNeasy UltraClean 96 Microbial kit (10196-4, Qiagen), and prepared for whole-genome sequencing using Nextera Tagmentation (Baym et al., 2015). Briefly, gDNA was brought to a concentration of 0.5 ng/mL in 1 mL volume. To each sample, 1.25 mL TD buffer, 0.125 mL TDE1 enzyme, and 0.125 mL nuclease-free water was added, and incubated at 55 C for 15 m. Then, 11.2 mL KAPA HiFi master mix was added to each tagmented sample. Indexed sequencing adaptors (5 mM each) were thawed, and 8.8 mL was added to each sample. Then, PCR was performed using the following protocol: 72 C for 3 m, 98 C for 5 m, 98 C for 10 s, 63 C for 30 s, 72 C for 30s, return to step 2 13 times, 72 C for 5 m, 4 C forever. PCR reactions were purified using AMPure XP beads according to the manufacturer’s protocol, and eluted in 60 mL resuspension buffer. Sequencing libraries were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Fisher Scientific), and pooled at equimolar concentrations for sequencing. 2 million 2x150bp sequencing reads were obtained per sample.

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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

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Manufacturer protocol

Download the product protocol from Qiagen for DNeasy UltraClean 96 Microbial Kit (384) below.

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