Jun 14, 2015
Qiagen MinElute Gel Extraction
- Michael Crone
Protocol Citation: Michael Crone 2015. Qiagen MinElute Gel Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.c2fybm
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 14, 2015
Last Modified: February 09, 2018
Protocol Integer ID: 807
Abstract
MinElute columns can also be used on any vacuum manifold with luer connectors (e.g., QIAvac 24 Plus or QIAvac 6S with Luer Adapters). The following protocol is designed to extract and purify DNA of 70 bp to 4 kb from standard or low-melt agarose gels in TAE or TBE buffer using vacuum-driven processing resulting in high end-concentrations of DNA. Up to 400 mg agarose can be processed per MinElute column.
Materials
STEP MATERIALS
Buffer QG
Sodium acetateP212121
Buffer QG
Sodium acetateP212121
Protocol materials
Buffer QG
Sodium acetateP212121
Buffer QG
Sodium acetateP212121
Buffer QG
Sodium acetateP212121
Before start
The yellow color of Buffer QG indicates a pH ≤7.5.
Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
Isopropanol (100%) and a heating block or water bath at 50°C are required.
All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.
Excision of Fragment
Excision of Fragment
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Solubilisation
Solubilisation
Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes Buffer QG.
Buffer QG
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel.
00:10:00
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
10 µL
Sodium acetateP212121
Add 1 gel volume of isopropanol to the sample and mix.
Binding
Binding
Place a QIAquick spin column into a provided 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 μl, load and spin/apply vacuum again.
00:01:00
Wash
Wash
If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 0.5 ml Buffer QG to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.
00:01:00
To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min.
Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt-ended ligation), let the column stand 2–5 min after addition of Buffer PE.
00:01:00
Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g (13,000 rpm) to remove residual wash buffer.
Elution
Elution
Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the center of the membrane for complete elution of bound DNA. The average eluate volume is 9 µl from 10 µl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
00:02:00
Analysis
Analysis
If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel. Loading dye contains 3 marker dyes (bromophenol blue, xylene cyanol, and orange G) that facilitate estimation of DNA migration distance and optimization of agarose gel run time. Refer to Table 3 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.