After DNA samples are run on an agarose gel, extraction involves four basic steps: identifying the fragments of interest, isolating the corresponding bands, isolating the DNA from those bands, and removing the accompanying salts and stain.
To begin, UV light is shone on the gel in order to illuminate all the ethidium bromide-stained DNA. Care must be taken to avoid exposing the DNA to mutagenic radiation for longer than absolutely necessary. The desired band is identified and physically removed with a cover slip or razor blade. The removed sliver of gel should contain the desired DNA inside. An alternative method, utilizing SYBR Safe DNA gel stain and blue-light illumination, avoids the DNA damage associated with ethidium bromide and UV light.
Detailed Gel Extraction Protocol Spin Columns
Spin column protocols like the QIAquick Spin Kit are made to extract DNA from about 100bp to 4kb.
Anything smaller or much bigger may have problems either going through or coming out of the spin filter respectively.
Most kits are designed to extract DNA from standard or low-melting temperature agarose gels made in TAE or TBE buffer, although they promise high yields a lot of DNA is lost in these preparations.
However, you get pretty pure target DNA for cloning in fairly high end-concentrations.
Most columns can process up to 400 mg agarose per column (including MinElute).
The yellow color of Buffer QG indicates a pH of 7.5.
Add ethanol (96â€“100%) to Buffer PE before use (see bottle label for volume).
Isopropanol (100%) and a heating block or water bath at 50Â°C are required.
All centrifugation steps are carried out at ?10,000 x g (~13,000 rpm) in a conventional table-top microcentrifuge.
3 M sodium acetate, pH 5.0, may be required.
Steps in the Gel Extraction Protocol
1) You have to excise the DNA fragment from the agarose gel using a sterile, clean, and sharp scalpel.
Then you place the gel slice into a 1.5-ml microcentrifuge tube and optionally store it in the fridge overnite. Use of the DNA is best when fresh because the ends of the DNA are exposed to DNAses and
UV light which can damage the DNA and prevent efficient ligation later of the sticky ends.
For gels stained with SYBR Safe DNA Gel Stain and illuminated with the Safe Imager Blue-Light Transilluminator, no special precautions are necessary.
For gels stained with ethidium bromide and visualized with UV, be sure to wear complete face and eye protection as well as a lab coat.
Minimize the size of the gel slice by removing extra agarose.
2) Weigh the gel slice, using an empty tube to tare the balance. Then weigh your gel slice with the tube.
3) Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 Âµl). For example, add 300 Âµl of Buffer QG to each 100 mg of gel. 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.
4) Incubate at 50Â°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2â€“3 min during the incubation.
NOTE: Make sure you solubilize the agarose completely. For >2% gels, increase incubation time.
5) 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 Âµl 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.
6) Add 1 gel volume of isopropanol to the sample and mix.
For example, if the agarose gel slice is 100 mg, add 100 Âµl isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield.
Do not centrifuge the sample at this stage.
7) 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.
8) The maximum volume of the column reservoir is 800 Âµl. For sample volumes of more than 800 Âµl, simply load and spin again.
9) Discard flow-through and place QIAquick column back in the same collection tube.
Collection tubes are re-used to reduce plastic waste.
9A) (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.
10) To wash, add 0.75 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 2â€“5 min after addition of Buffer PE, before centrifuging.
11) Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 13,000 rpm (~17,900 x g).
NOTE: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12) Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13) To elute the DNA from the column, add 50 Âµl of Buffer EB (10 mM TrisÂ·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 Âµl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
NOTE: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 Âµl from 50 Âµl elution buffer volume, and 28 Âµl from 30 Âµl.
Gel Extraction Protocol Notes
The gels can keep for up to 1 week without significant problems.
DNA can be stored in TE at 4C for up to 1 week. For longer storage I prefer to keep it at -20C.
To analyze the extraction, you can run 3uL on an agarose gel to verify recovery and amount for ligation.
If you use your own water for ligation check the pH first!
Do an ethanol precipitation to clean and concentrate the vector after purification to remove agarose. Agarose is known to inhibit ligation reactions.
If you are getting low yields, then prepare multiple reactions to give you a total volume of up to 200ul. Then add the vector and ethanol precipitate the two together. Resuspend the final DNA pellet in 10ul for the ligation.
Electroelution Dialysis DNA Gel Extraction Protocol
Run 20-50ug of digested DNA in GTG grade agarose using TBE or TAE buffer.
Excise band from the agarose gel using a clean ethanol wiped razor blade.
Trim the gel strip to fit into the smallest size dialysis membrane (Spectra/pore 4MW cutoff 12-14,000).
Take a prepared hydrated length of dialysis tubing about 4 cm longer than the length of the gel strip. *Close one end with a dialysis bag clip.
Place 0.5-1.0 ml of 0.5X TBE into the tubing (this will allow the gel strip to slide in smoothly. You can squeeze the liquid up to the top of the tubing while dangling the gel strip to accomplish this).
Apply the second clip. In so doing you may remove up to 1/2 of the liquid from the tubing, taking care that there are no air bubbles.
Orient the tubing in a gel box so that it is exactly parallel to the electrodes and perpendicular to the electrical field in the buffer. Cover the tubing with 0.5X TBE. Run the fragment out of the strip and on to the wall of the tubing at 30-50 mA for about 30 minutes. Monitor progress using a hand-held long wavelength UV illuminator in the darkened room. Stop when the fragment has completely lined up on the inside of the dialysis tubing.
Reverse the electrodes and run the power supply at the same settings for 0.5-2.0 minutes. Monitor progress again as described above. You should see the EtBr-stained DNA move off of the inside wall of the tubing.
Remove the top clip and cut off any excess dialysis tubing. Draw off the DNA-containing liquid, trimming the tubing if necessary.
Add 0.1 volume of 3M NaAc and 2-3 volumes of absolute ethanol. Precipitate for 1 hour or more.
Resuspend DNA in 40-80 ul injection buffer (10mMTris/0.1mMEDTA, pH 7.4) and pass it through an ion exchange column (e.g., Schleicher & Shuell Elutip columns) according to the manufacturerâ€™s instructions
Important Tips for Gel Extraction Protocol
Ensure that you pipette the elution buffer onto the center of the spin column membrane for high efficiency elution of bound DNA. The DNA needs to be rehydrated and dissolved by the elution buffer, so if you add the buffer to the sides you are missing most of the DNA that is in the middle of the column.
Elution efficiency is dependent on the final 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 â€“20Â°C as DNA may degrade in the absence of a buffering agent.
Also if you are cloning, be wary that EDTA present in your buffers and other factors may inhibit subsequent ligation reactions. The purified DNA can also be eluted in TE (10 mM TrisÂ·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
Other Gel Extraction Strategies
Several other strategies for isolating and cleaning the DNA fragment of interest exist.
Spin Column Extraction
Gel extraction kits are available from several major biotech manufacturers for a final cost of approximately 1â€“2 US$ per sample. Protocols included in these kits generally call for the dissolution of the gel-slice in 3 volumes of chaotropic agent at 50 degrees C, followed by application of the solution to a spin-column (the DNA remains in the column), a 70% ethanol wash (the DNA remains in the column, salt and impurities are washed out), and elution of the DNA in a small volume (30ÂµL) of water or buffer.
The gel fragment is placed in a dialysis tube that is permeable to fluids but impermeable to molecules the size of DNA, thus preventing the DNA from passing through the membrane when soaked in TE buffer. An electric field is established around the tubing (in a way similar to gel electrophresis) long enough so that the DNA is removed from the gel but remains in the tube. The tube solution can then be pipetted out and will contain the desired DNA with minimal background.
The traditional method of gel extraction involves creating a folded pocket of Parafilm wax paper and placing the agarose fragment inside. The agarose is physically compressed with a finger into a corner of the pocket, partially liquifying the gel and its contents. The liquid droplets can then be directed out of the pocket onto an exterior piece of Parafilm, where they are pipetted into a small tube. A butanol extraction removes the ethidium bromide stain, followed by a phenol/chloroform extraction of the cleaned DNA fragment.
The disadvantage of gel isolation is that background can only be removed if it can be physically identified using the UV light. If two bands are very close together, it can be hard to separate them without some contamination. In order to clearly identify the band of interest, further restriction digests may be necessary. Restriction sites unique to unwanted bands of similar size can aid in breaking up these potential contaminants.
Recommended Gel Extraction Kits
QIAquick Spin Kit
â†‘ Quest: An Invitrogen Publication for Discovery Vol. 4, Issue 2, pp. 44â€“45 (2007).