QIAprep 96 Turbo BioRobot Kit (4)

DNA isolation / purification Bacteria - Gram negative E.coli

Experiment
DNA isolation / purification Bacteria - Gram negative E.coli
Product
QIAprep 96 Turbo BioRobot Kit (4) from Qiagen
Manufacturer
Qiagen

Protocol tips

Publication protocol

Screening of genes encoding glycosyl hydrolase from environmental DNA: To amplify partial fragments of genes encoding GHF10, 10–250 ng of each DNA isolated from digestive tracts of horses or termites were added to 50 μl of EX Taq Buffer (TaKaTa‐Bio) containing 0.2 mM each of dNTPs, 1 μM each of primers, XYN‐Fw and XYN‐Rv (Table 3), and 5 U of Ex Taq HotStart version (TaKaRa‐Bio). Polymerase chain reaction was performed with the following cycling programme: 1 cycle of 30 s at 95°C; 30 cycles of 30 s at 95°C, 30 s at 55°C and 15 s at 72°C; and 7 min of final extension at 72°C. The PCR product was purified, ligated to the pCR®4‐TOPO® Vector with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol, and used to transform ECOSTM‐competent E. coli DH5α cells (Nippongene). Transformants were plated on LB agar containing 50 μg ml−1 ampicillin. Colonies were picked up, suspended in 1.3 ml of LB medium in the wells of a deep‐well 96‐well plate (Qiagen, Hilden, Germany) and grown at 37°C for 20 h. Cells in the cultures were harvested, and plasmid DNA was isolated by using a QIAprep® 96 Turbo BioRobot Kit (Qiagen) and a Qiagen Biorobot 8000 (Qiagen). DNA sequencing was performed by using M13‐reverse and forward primers and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), and analysed by using an ABI3130 Genetic Analyser. Sequence data were assembled by using ATGC software (Applied Biosystems).

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Discussion

Discussion

5 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

5 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 QIAprep 96 Turbo BioRobot Kit (4) below.

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