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Team:Newcastle/23 July 2010
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====Materials Required==== | ====Materials Required==== | ||
| - | *Cells grown from yesterday | + | * Cells grown from yesterday |
| - | *Centrifuge | + | * Centrifuge |
| - | *pipette | + | * pipette |
| - | *lysozyme | + | * lysozyme |
| - | *Cell lysis solution | + | * Cell lysis solution |
| - | * | + | * RNase solution |
| - | *protein orecipitation solution | + | * protein orecipitation solution |
| - | *ice | + | * ice |
| - | *isopropanol | + | * isopropanol |
| - | *ethanol | + | * ethanol |
====Procedure==== | ====Procedure==== | ||
=====Cell lysis===== | =====Cell lysis===== | ||
| - | #Pellet cells by centrifugation at 3600rpm for 10 minutes | + | # Pellet cells by centrifugation at 3600rpm for 10 minutes |
| - | #Pour off supernatant | + | # 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 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 | + | # Add 25microlitres of lysozyme and invert 25 times |
| - | #Incubate for 30minutes at 37°C inverting occasionally | + | # Incubate for 30minutes at 37°C inverting occasionally |
| - | #Centrifuge at 13000rpm for 10minutes to pellet the cells then remove the supernatant | + | # 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 | + | # 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 | + | # Heat sample for 30 mins mix every 5-10 mins |
=====RNase treatment===== | =====RNase treatment===== | ||
| - | #Add 3 microlitres of RNAse A solution to the cell lysate | + | # Add 3 microlitres of RNAse A solution to the cell lysate |
| - | #Mix by inverting 25 times and incubate at 37°C for 60 minutes | + | # 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. | ||
| + | # | ||
Revision as of 13:41, 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.
