QIAquick Gel Extraction Kit, Cat# 28704 (50 reactions) or 28706 (250 reactions)
DNA fragment(s) seperated on agarose gel
1.5 ml centrifuge tubes
Thermomixer
Bench-top centrifuge
Analytical balance
Pipetter and tips
Scalpel
UV light platform
UV protection Mask
Gloves
Procedure
(partially Adapted from Qiagen product manual)
Weigh the number of needed microcentrifuge tubes and label.
Locate the DNA fragment on agarose gel on/under UV light.
Cut the DNA fragment with a clean, sharp scalpel and put in a microcentrifuge tube. Minimize the size of the gel slice by removing extra agarose.
Weigh the gel slice in the tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl). For >2% agarose gels, add 6 volumes of Buffer QG.
The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.
Incubate at 50°C on Thermomixer for 10 min or more and vortex 10 seconds every 2-3 min. Make sure the agarose gel is completely solubilized.
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 of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
Add 1 gel volume of isopropanol to the sample and mix. Do not centrifuge the sample at this stage.
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.
The maximum volume of the column reservoir is 800 µl. For sample volumes of more than 800 µl, simply load and spin again.
Discard flow-through and place QIAquick column back in the same collection tube.
(Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription or microinjection.
To wash, add 0.7 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after addition of Buffer PE, before centrifuging.
Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 13,000 rpm (~17,900 x g).
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 30-50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min.