IGEM:Yale/2010/Protocols/gel extraction

Gel Extraction Protocols

Unless indicated otherwise, all gel extractions were accomplished using a QIAquick Gel Extraction Kit. These protocols are taken from the QIAQuick Spin Handbook, November 2006 edition.

Vacuum Manifold Protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg is approximately 100 uL). If the color of the mixture is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
3. Incubate at 50 degrees Celsius for 10 minutes (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 minutes during the incubation. For gels greater than 2% agarose, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow ( similar to the Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
6. To bind DNA, pipet the sample onto the QIAquick column and apply vacuum. After the sample has passed through the column, switch off the vacuum source.
7. Add 0.5 ml of Buffer Q to QIAquick column and apply vacuum.
8. To wash, add 0.75 ml of Buffer PE to QIAquick column and apply vacuum.
9. Transfer QIAquick column to provided 2 mL collection tube. Centrifuge for 1 min at 17,900 times g (13,000 rpm).
10. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.
11. To elute DNA, add 50 uL of Buffer Ed (10 mM Tris*Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 minute. Alternatively, for increased DNA concentration, add 30 uL elution buffer to the center of the QIAquick membrane, let the column stand for 1 minute, and then centrifuge for 1 minute.
12. 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.

Microcentrifuge Protocol

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg is approximately 100 uL). If the color of the mixture is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
3. Incubate at 50 degrees Celsius for 10 minutes (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 minutes during the incubation. For gels greater than 2% agarose, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow ( similar to the Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
5. Add 1 gel volume of isopropanol to the sample and mix.
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 minute. Discard flow-through and place the QIAquick column back into the same tube. The maximum volume of the column reservoir is 800 uL. For sample volumes of more than 800 uL, simply load and spin again.
8. Recommended: Add 0.5 mL of buffer QG to QIAquick column and centrifuge for 1 minute. Discard flow-through and place the QIAquick column back into the same tube. This step is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription, or microinjection.
9. To wash, add 0.75 mL of Buffer Pe to QIAquick column and centrifuge for 1 minute. Discard flow-through and place the QIAquick column back into the same tube. 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 minutes after addition of Buffer PE, before centrifuging.
10. Centrifuge the column in a 2 mL collection tube (provided) for 1 min at 17,900 times g (13,000 rpm).
11. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.
12. To elute DNA, add 50 uL of Buffer Ed (10 mM Tris*Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 minute. Alternatively, for increased DNA concentration, add 30 uL elution buffer to the center of the QIAquick membrane, let the column stand for 1 minute, and then centrifuge for 1 minute.
13. 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.