Team:Cambridge/Protocols/DNA Gel Extraction

Protocol Name

If a DNA sample is to be treated further, once the size of a PCR product or cellular nucleic acid isolate has been determined, it must first be extracted from the agarose.

Theory

How it works The initial extraction from the gel was made with special electrophoresis band cutting tips which fit on a Gilson pipette and suck up approximately 100mg of agarose containing the DNA sample.

The extracted gels were subjected to the QIA Gel Extraction protocol detailed below. These rely on extraction columns, tubes that bind DNA strongly in the presence of one buffer, and release it in the presence of another.

Practice

How to do it in the lab QIAquick Gel Extraction Kit Protocol using a microcentrifuge This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8). For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction, mix, and proceed with step 6 of the protocol. Alternatively, use the MinElute Reaction Cleanup Kit. Important points before starting

■ 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).

■ All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature. Procedure

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl). For example, add 300 μl 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.

3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.

4. 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 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.

5. Add 1 gel volume of isopropanol to the sample and mix. For example, if the agarose gel slice is 100 mg, add 100 μl 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. QIAquick Spin Handbook 03/2008 25

6. Place a QIAquick spin column in a provided 2 ml collection tube.

7. 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.

8. Discard flow-through and place QIAquick column back in the same collection tube. Collection tubes are reused to reduce plastic waste.

9. Recommended: 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 2–5 min after addition of Buffer PE, before centrifuging.

11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm). IMPORTANT: 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 DNA, add 50 μl of 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 for 1 min. 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 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 (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

14. 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.

Safety

The safety implication of the procedure.