Sep 17, 2015
Qiagen QIAquick Gel Extraction Kit (28704 and 28706), centrifuge processing
Forked from Qiagen QIAquick PCR Purification Kit Protocol
Protocol Citation: Harold Bien: Qiagen QIAquick Gel Extraction Kit (28704 and 28706), centrifuge processing. protocols.io https://dx.doi.org/10.17504/protocols.io.dt96r5
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: September 17, 2015
Last Modified: March 28, 2018
Protocol Integer ID: 1633
Abstract
The Qiagen QIAquick Gel Extraction kit (catalog #28704 and 28706) are for extraction of DNA fragments (70 bp – 10 kb) from standard or low-melt agarose gels in TAE buffer (Tris·acetate/EDTA) or TBE buffer (Tris·borate/ EDTA) and DNA cleanup from enzymatic reactions. The kit is suitable for purification of up to 10μg of DNA (70bp to 10kb).
Materials
STEP MATERIALS
Buffer PE
Buffer PE
Protocol materials
Buffer PE
Buffer PE
Buffer PE
Before start
Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge at room temperature (15–25°C).
Add 1:250 volume pH Indicator I to Buffer PB (i.e., add 120 μl pH Indicator I to 30 ml Buffer PB or add 600 μl pH Indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH Indicator I indicates a pH of ≤ 7.5. Add pH Indicator I to entire buffer contents. Do not add pH Indicator I to buffer aliquots. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH Indicator I.
Isopropanol (100%) and a heating block or water bath at 50°C are required
Extraction
Extraction
Excise DNA fragment from agarose gel with clean, sharp scalpel
Weigh the gel slice in a colorless tube. Maximum amount of gel per column is 400mg (about 0.4cm3)
Add 3 volumes Buffer QG to 1 volume of gel (100mg gel ~ 100μL). For >2% agarose gels, add 6 volumes Buffer QG
3 µL
Incubate at 50°C for 10 minutes (or until gel slice has completely dissolved). [Optional]: Vortex every 2-3 min to help dissolve gel.
00:10:00
Check color of mixture to ensure it is yellow, similar to Buffer QG without dissolve agarose. If color is orange or violet, add 10μL of 3M sodium acetate, pH 5.0, and mix.
For purifying <500bp or >4kb, add 1 volume isopropanol to increase yield and mix
1 µL
Binding
Binding
Place a QIAquick spin column in a provided 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min then discard flow through. The maximum volume is 800μL. For larger volumes, load and spin again.
00:01:00
Return QIAquick column to collection tube
Wash
Wash
If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500μL Buffer QG to the QIAquick column and centrifuge for one minute.
00:01:00
Discard flow through and return column to collection tube
To wash, add 750 μl Buffer PE to the QIAquick column
Buffer PE
If DNA will be used for salt-sensitive applications (e.g. sequencing, blunt-ended ligation), let column stand for 2-5 minutes
00:05:00
Centrifuge for 1 minute
00:01:00
Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
00:01:00
Elution
Elution
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer.
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.
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 2 (page 15) to identify the dyes according to migration distance and agarose gel percentage and type.