Team:Newcastle/27 July 2010

Aims of genomic DNA extraction experiment

The aim of today's experiment is to extract genomic DNA from Bacillus subtilis strain 3610 and 168,genes from which will be needed for the swarming biobrick and rocF biobrick.

Materials Required

• Cells grown from yesterday
• Centrifuge
• pipette
• lysozyme
• Cell lysis solution
• RNase solution
• protein precipitation solution
• ice
• isopropanol
• ethanol

Procedure

Cell lysis

1. Pellet cells by centrifugation at 3600rpm for 10 minutes.
2. Pour off supernatant.
3. Add 0.5ml of cell suspension solution, gently pipet up and down to resuspend and transfer to 1.5 ml eppendorf tube.
4. Add 25microlitres of lysozyme and invert 25 times.
5. Incubate for 30 minutes at 37°C inverting occasionally.
6. Centrifuge at 13000rpm for 10minutes to pellet the cells then remove the supernatant.
7. Add 0.5ml of cell lysis solution to the cell pellet and gently pipet up and down to lyse the cells.
8. Heat sample for 30 minutes mix every 5-10 minutes.

RNase treatment

1. Add 3 microlitres of RNase A solution to the cell lysate
2. Mix by inverting 25 times and incubate at 37°C for 60 minutes

Protein precipitation

1. Cool samples on ice.
2. Add 0.5 ml protein precipitation solution to each tube.
3. Vortex vigorously at high speed for 20 seconds to miux the protein precipitation solution uniformly with the cell lysate. Place samples on ice for 5 minutes.
4. Centrifuge at 13000 rpm for 30 seconds or until the precipitated proteins form a tight pellet.

DNA precipitation

1. Pour the supernatant containing the DNA into a clean eppendorf tube. (The samples may be kept at -20°C overnight at this stage.)
2. Add 0.5ml isopropanol to each tube.
3. Mix by inverting gently for 50 times.
4. Centrifuge at 13000 rpm for 1 minute. The DNA should be visible as a small white pellet.
5. Pour off the supernatant and drain the tube on clean absorbent paper. Add 0.5 ml 70% ethanol and invert tube several times to wash the DNA.
6. Centrifuge at 13000 rpm for 1 minute. Carefully pour off the ethanol.
7. Drain the tubes on clean absorbent paper. Allow to air dry for 10-15 minutes.

DNA hydration

1. Add 100 µl DNA hydration solution to each tube.
2. Rehydrate DNA by incubating the sample for 1 hour at 65°C and overnight at room temperature. Tap the tube periodically to aid in dispersing the DNA.
3. For storage, centrifuge briefly and store at -20°C.

Discussion

At the end of the DNA rpecipitation step, we did not observe any small white pellet.

Conclusion

If the experiment has failed the experiment will be redone on Monday, 26th July, 2010.