Team:UNICAMP-Brazil/Notebooks/August 7

Genomic DNA Extraction for Hly Operon Manipulation

We performed the genomic DNA Extraction from four pathogenic E. coli hly+ strains. We used the Aubusel 1998 protocol with modifications. The protocol is followed below:

1- Grow bacterial strain in 4ml of LB medium overnight.

2- Centrifuge 2ml of the medium for 5 minutes at 6000rpm to pellet the cells

3- Resuspend the pellet in 400µl TE buffer by repeated pipetting. Add 30µl SDS 10% and 2,5µl 20mg/ml proteinase K. Mix thoroughly and incubate for 30 minutes at 37°C.

4- Add 100µl NaCL 5M. Mix thoroughly.

5- Add 100µl CTAB/NaCL solution ( 0,8G/l CTAB, 0,4g/l NaCl). Incubate 10 min at 65°C.

6- Add 750µl chloroform/isoamyl alcohol (24:1). Mix thoroughly and centrifuge for 10 min at 1200rpm. The original protocol uses phenol in this phase, but we are avoiding using this substance due its risks to our health and the environment.

7- Recover aqueous supernatant to a new tube, leave the interface behind. Add 600µl isopropanol at -20°C to precipitate the nucleic acids. Incubate at room temperature for 30min.

8- Centrifuge for 30 min at 12000rpm, discard the liquid maintaining the pellet in the tube. Wash the pellet 2x with ethanol 70% at -20°C.

9- Let the pellet dry at room temperature and then resuspend with 50µl H2O or TE.