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From 2010.igem.org

Gram-positive Bacteria Chromosomal DNA Extraction Protocol

Contents 1 Puregene DNA isolation 2 Cell Lysis 3 RNase Treatment 4 Protein Precipitation

Puregene DNA isolation

1. Grow overnight 7.5ml cultures in THYB containing 30μg/ml hyaluronidase.

Cell Lysis

1. Cells go through 3600 x g centrifugation for 15 minutes to pellet.
2. The supernatant is poured away.
3. 0.5ml of cell suspension solution is added to the eppendorf tube. The solution in the tube is pipetted up and down to resuspend cells. The solution is then transferred to another 1.5ml tube.
4. 50μl of lysozyme and 7.5μl of lytic enzyme solution is added into the tube. The tube is then inverted 25 times to mix the solution.
5. We then incubate it at 37°C for 30 minutes in order to digest the cell walls. The tubes are inverted occasionally during incubation.
6. To pellet the cells, we centrifuge it for 10 minutes and poured away the supernatant after that.
7. 0.5ml of cell lysis solution is added to the pelleted cells and the solution is pipetted up and down in order to lyse the cells. The samples are then heated for about 5 minutes at 80°C.

RNase Treatment

1. 6μl of RNase A solution is added into the cell lysate.
2. The tubes are inverted 25 times in order to allow the solutions to mix. It is then incubated at 60minutes at 37°C.

Protein Precipitation

1. Samples are cooled on ice.