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 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

Safety

The safety implication of the procedure.