Team:Newcastle/23 July 2010
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Contents |
Aims of the experiment
The aim of today's experiment is to extract genomic DNA from Bacillus subtilis strain 3610,genes from which will be needed for the swarming biobrick.
Materials Required
- Cells grown from yesterday
- Centrifuge
- pipette
- lysozyme
- Cell lysis solution
- RNase solution
- protein orecipitation solution
- ice
- isopropanol
- ethanol
Procedure
Cell lysis
- Pellet cells by centrifugation at 3600rpm for 10 minutes
- Pour off supernatant
- Add 0.5ml of cell suspension solution, gently pipet up and down to resuspend and transfer to 1.5ml eppendorf tube
- Add 25microlitres of lysozyme and invert 25 times
- Incubate for 30minutes at 37°C inverting occasionally
- Centrifuge at 13000rpm for 10minutes to pellet the cells then remove the supernatant
- Add 0.5ml of cell lysis solution to the cell pellet and gently pipet up and down to lyse the cells
- Heat sample for 30 mins mix every 5-10 mins
RNase treatment
- Add 3 microlitres of RNAse A solution to the cell lysate
- Mix by inverting 25 times and incubate at 37°C for 60 minutes
Protein precipitation
- Cool samples on ice.
- Add 0.5 ml protein precipitation solution to each tube.
- 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.
- Centrifuge at 1300 rpm for 30 seconds or until the precipitated proteins form a tight pellet.
DNA precipitation
- Pour the supernatant containing the DNA into a clean eppendorf tube. (The samples may be kept at -20°C overnight at this stage.)
- Add 0.5ml isopropanol to each tube.
- Mix by inverting gently for 50 times.
- Centrifuge at 1300 rpm for 1 minute. The DNA should be visible as a small white pellet.
- 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.
- Centrifuge at 1300 rpm for 1 minute. Carefully pour off the ethanol.
- Drain the tubes on clean absorbent paper. Allow to air dry for 10-15 minutes.
DNA hydration
- Add 100 µl DNA hydration solution to each tube.
- 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.
- For storage, centrifuge briefly and store at -20°C.