Team:Cambridge/Protocols/Gel Extraction of DNA

Gel Extraction of DNA (Spin Column Extraction)

A technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis

Theory

Gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process forms the basis for rudimentary genetic engineering.

Practice

We used the QIAgen quick gel extraction kit, the protocol is adapted from the QIAgen gel extraction protocol [http://openwetware.org/images/5/5e/QIAquick_Gel_Extraction_Kit_Protocol.pdf QIAquick Gel Extraction Kit Protocol].

• Extraction From Gel

1. The gel is exposed to blue-light to illuminate the DNA fragments(stained by SYBR safe dye).
2. The desired DNA band is identified and physically removed with a rectangular tipped pipette. The removed slice of gel contains the desired DNA.

• DNA Purification

1. Add 3 volumes of buffer QG to 1 volume of gel. E.g.add 300 μl of Buffer QG to each slice of gel (volume of the slice is 100 μl, and weighs approximately 100 mg).
2. Incubate at 50°C for 10 min (or until fully dissolved). To aid solvation, mix by vortexing the tube every 2–3 min during the incubation.
3. After the gel slice has dissolved fully, check that the colour of the mixture is yellow (similar to Buffer QG without dissolved agarose).
4. Add 1 gel volume of isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 ml collection tube.
6. Apply each sample of DNA fragment to a QIAquick column, and centrifuge for 1 min at 13000 rpm.
7. Discard flow-through and place QIAquick column back in the same collection tube. (Optional)
8. (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.
9. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
   • If this step is omitted, follow Rescue Precipitation of DNA
10. Discard the flow-through and centrifuge the QIAquick 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.
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H₂O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13000rpm. 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.

Safety

Since the agarose gel has been dyed with SYBR Safe, everything that could have come into contact with it needs to be treated as toxic waste, including gloves (which must be worn at all times during the whole procedure).