Notebook – Protocol – 4. DNA Purification

Spin column based purification was used. The kit is Gel/PCR extraction kit (Nippon Genetics). Protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge. All steps were done at room temperature.

DNA Purification from Liquid Samples

1. Into a clean, sterilized 0.5 ul polypropylene tube, add PCR products : buffer GP1 = 1 : 5 (e.g. 40 ul : 200 ul). Vortexing.

2. Load the samples onto the spin column equipped with collection tube. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.

3. Add 600 ul of GP2. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.

4. Centrifuge for 6,000 rpm; 5 minutes to dry the column.

5. Replace collecting tube with clean, sterilized 1.5 ml size polypropylene tube.

6. Add 30 ul of GP3. Incubate 2 minutes at room temperature. Centrifuge for 6,000 rpm; 5 minutes.

7. Obtain the DNA as the flowthrough.

DNA Purification from Agarose Gel

1. Into a clean, sterlized 1.5 ul polypropylene tube, add cutted gel containing DNA of interest : buffer GP1 = < 300 mg : 500 ul. Vortexing. Incubate at 55 oC; 10-15 minutes. Invert the tube.

2. Load the samples onto the spin column equipped with collection tube. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.

3. Add 600 ul of GP2. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.

4. Centrifuge for 6,000 rpm; 5 minutes to dry the column.

5. Replace collecting tube with clean, sterilized 1.5 ml size polypropylene tube

6. Add 30 ul of GP3. Incubate 2 minutes at room temperature. Centrifuge for 6,000 rpm; 5 minutes.

7. Obtain the DNA as the flowthrough.

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