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 is the basis for rudimentary genetic engineering.

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, blue-light illumination is shone on the gel in order to illuminate all the DNA fragments(stained by SYBR safe dye). The desired band is identified and physically removed with pipette with a special rectangular pipette tip. The removed slice of gel should contain the desired DNA inside.

Practice

Used 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].

Step 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 weight of the slice is approximately 100 mg).

Step 2

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.

Step 3

After the gel slice has dissolved completely, check that the color of the mixture is

yellow (similar to Buffer QG without dissolved agarose).

Step 4

Add 1 gel volume of isopropanol to the sample and mix.

Step 5

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

Step 6

apply each sample of DNA fragment to a QIAquick column, and centrifuge for 1 min at 13000 rpm.

Step 7

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

Step 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.

Step 9

To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.

Step 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.

Step 11

Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

Step 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

The safety implication of the procedure.