Team:UNICAMP-Brazil/Protocols

Protocols

Exemplos: (Vá em editar, e troque Protocolo_1 e Protocolo 1 pelo título correto (coloque o número na frente)

1 Genomic DNA Extraction

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.

2 Mini-Prep

This procedure is used to extract plasmid DNA from bacterial cell suspensions and is based on the alkaline lysis procedure developed by Birnboim and Doly (Nucleic Acids Research 7:1513, 1979).

Procedure

1. Gently swirl the contents of the culture tube to resuspend the cells.

2. Label 1.5 mL tubes and pipet 1500 uL of the cell suspension into each tube.

3. Close the caps and place the tubes in a centrifuge and spin at maximum speed for 2 minutes.

4. Withdraw and discard the supernatant in a waste container.

5. Add 300 uL of Buffer 1 (50 mM Tris-HCl, 10 mM EDTA, 100 ug/mL RNase A, pH 8.0 ) to each tube and resuspend the cells by vortexing. It's very important that the cell suspension is homogenous and no clumps are visible.

6. Add 300 uL of Buffer 2 (1% SDS, 0.2 M NaOH ) to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. DO NOT VORTEX since the chromosomal DNA released from the broken cells could be sheared into small fragments and contaminate your plasmid prep.

7. Let tubes stand on ambient temperature for 5 minutes.

8. Add 300 uL of ice-cold Buffer 3 (3.0 M Potassium Acetate, pH 5.5 ) to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. A white precipitate will form.

9. Let tubes stand on ambient temperature for 15 minutes.

10. Place the tubes in a centrifuge (balanced) and spin at maximum speed for 15 minutes. The precipitate will pellet along the side of the tube.

11. Transfer the supernatants into clean 1.5 mL tubes, being careful not to pick up any of the precipitate. Discard the tubes with the precipitate and KEEP the tubes with the supernatant.

12. To each tube of supernatant add 400 uL of isopropanol to precipitate the nucleic acids. Close the caps and mix vigorously, place them in a centrifuge (balanced) and spin at maximum speed for 10 minutes. This step pellets the nucleic acids but if you leave it around too long, proteins remaining in solution will begin to precipitate as well.

13. Plasmid DNA pellet may be difficult to see. Carefully remove and discard the supernatant. The pellet is usually visible at this point. If not, do not despair. It may be too small to see but there is probably enough DNA there.

14. Add 600 uL of absolute ethanol to each tube and mix by inversion several times.

15. Spin the tubes at maximum speed in a centrifuge for 5 minutes.

16. Carefully remove and discard the supernatant. Try to get as much out as possible without dislodging the pellet of plasmid DNA.

17. Place the tubes in the fume hood with the caps open for 15-20 minutes to dry off the last traces of ethanol.

18. When the ethanol is gone (you can check this by smelling the tube) add 20 uL of MiliQ water to dissolve the pellet. Pipet the 20 uL in and out, up the side of the tube to ensure that all of the plasmid DNA comes into contact with the water.

19. Pool the 20 uL solutions into one labeled tube and store it in the freezer