MinElute PCR Purification Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.	Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.
Protocol tips
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Publication protocol

The protocol is based on [29,30]. Between 15 and 30 μl of DNA extract was used in a 50 μl blunting reaction with a final concentration of 1× buffer Tango, 100 μM each dNTP, 1 mM ATP, 25 U T4 polynucleotide kinase and 5 U T4 DNA polymerase (all reagents from Thermo Scientific Fermentas Molecular Biology Solutions Waltham, MA). After incubation at 25°C for 15 min and 12°C for 5 min, the reaction was cleaned up with the MinElute PCR purification kit (Qiagen, Hilden, Germany) by adding 250 μl of buffer PB to each reaction and applying the mixture to the MinElute column and centrifuging. Two washing steps with buffer PE were performed and after a dry-spin, DNA was eluted in 18 μl 10 mM Tris–HCl.

Each DNA extract was assigned a unique barcode combination and within each batch no single barcode was used more than once. For each library, 1 μl of a barcoded, partially double-stranded P5-adapter (10 μM) and 1 μl of a barcoded, partially double-stranded P7-adapter (10 μM) were added to the blunted DNA and mixed before adding the ligation mix to bring adapters and DNA into close proximity (final concentration 0.25 μM for each barcoded adapter). The concentrations in the 40 μl final ligation reactions were as follows: 1× T4 DNA ligase buffer, 5% PEG-4000, 5 U T4 DNA ligase (all reagents from Thermo Scientific Fermentas Molecular Biology Solutions, Waltham, MA). After mixing and a quick spin, the reaction was incubated for 30 min at room temperature. MinElute clean-up was performed by adding 200 μl buffer PB to each finished ligation reaction, washing with PE buffer twice, and eluting in 20 μl 10 mM Tris–HCl.

The fill-in reaction was performed in a final volume of 40 μl with 1× ThermoPol buffer (New England Biolabs (NEB), Ipswich, MA), 250 μM each dNTP (Thermo Scientific Fermentas Molecular Biology Solutions) and 16 U Bst polymerase, large fragment (NEB) and incubated at 37°C for 20 min followed by a heat-inactivation at 80°C for 20 min [29,30]. The entire 40 μl heat-inactivated fill-in reaction was used in the PCR that finished the library preparation. A total of 400 μl PCR mix (divided into four to eight reactions) per sample was prepared with the following final concentration: 1× Pfu Turbo Cx reaction buffer, 20U Pfu Turbo Cx Hotstart DNA Polymerase (both Agilent Technologies, Santa Clara, CA), 200 μM each dNTP (Thermo Scientific Fermentas Molecular Biology Solutions) and 400 nM of each of the two primers (PreHyb-F, PreHyb-R, sequences in the electronic supplementary material, table S1) that do not extend the adapter sites but keep them truncated (short). After an initial denaturation and activation of the polymerase at 95°C for 2 min, 30 cycles at 95°C for 30 s, 55°C for 30 s and 72°C for 1 min, we performed a final extension at 72°C for 5 min. Following PCR, the product was cleaned up with the MinElute PCR purification kit by adding 2 ml buffer PB to the 400 μl PCR and distributing the mix onto two MinElute columns. After washing the silica matrix twice with PE buffer and a dry-spin, each column was eluted in 25 μl 1× TE (with 0.05% Tween-20), resulting in 50 μl final library.

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Manufacturer protocol

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