Molecular Biology Protocols Marine DNA Sequencing and Analysis Center Mount Desert Island Biological Laboratory

Image courtesy of Qiagen Last update January 13, 2006.  Send comments to dtowle@mdibl.org

8. QiaQuick Gel Extraction Protocol

Preparing PCR Products for Direct Sequencing

If you discover that your PCR experiment has amplified your sequence of interest, first dance a little, then gel purify all of your sample in aliquots to prepare sufficient template for sequencing, setting up enough lanes in a second gel to accommodate your sample(s). For both the original gel and the second gel, cut out the desired bands according to the following procedure:

MinElute Gel Extraction Kit Protocol Using a Microcentrifuge (Courtesy of the Qiagen website http://www.qiagen.com/)

This 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 resulting in high end-concentrations of DNA. Up to 400 mg agarose can be processed per MinElute column.

Notes: • 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 ≥10,000 x g (~13,000 rpm) in a conventional table-top microcentrifuge.

• 3 M sodium acetate, pH 5.0, may be required.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel and place the gel slice into a 1.5-ml microcentrifuge tube. 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. 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. Note: Qiagen states that the maximum amount of gel slice per spin column is 400 mg and recommends for gel slices >400 mg to use more than one MinElute column. However, we have found that we can load DNA from up to 1200 mg of gel onto one column to produce a concentrated product.

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).

Note: 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 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 by inverting the tube several times. For example, if the agarose gel slice is 100 mg, add 100 µl isopropanol. Do not centrifuge the sample at this stage.

6. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.

7. To bind DNA, apply the sample to the MinElute column, and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column. 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 the flow-through and place the MinElute column back in the same collection tube.

9. Add 500 µl of Buffer QG to the spin column and centrifuge for 1 min.

10. Discard the flow-through and place the MinElute column back in the same collection tube.

11. To wash, add 750 µl of Buffer PE to the MinElute 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.

12. Discard the flow-through and centrifuge the MinElute column for an additional 1 min at ≥10,000 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.

13. Place the MinElute column into a clean 1.5 ml microcentrifuge tube.

14. To elute DNA, add 10 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) 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.