"
Team:Newcastle/23 July 2010
From 2010.igem.org
(Difference between revisions)
Shethharsh08 (Talk | contribs) |
Shethharsh08 (Talk | contribs) |
||
| Line 37: | Line 37: | ||
# Add 0.5 ml protein precipitation solution to each tube. | # 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. | # 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===== | ||
Revision as of 13:42, 23 July 2010
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.
