Team:Newcastle/DNA extraction
From 2010.igem.org
(Difference between revisions)
(New page: ====Materials Required==== * Cells grown from yesterday * Centrifuge * pipette * lysozyme * Cell lysis solution * RNase solution * protein precipitation solution * ice * isopropanol * eth...) |
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- | ====Materials Required | + | {{Team:Newcastle/mainbanner}} |
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+ | =DNA extraction= | ||
+ | |||
+ | ==Materials Required== | ||
* Cells grown from yesterday | * Cells grown from yesterday | ||
* Centrifuge | * Centrifuge | ||
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* ethanol | * ethanol | ||
- | == | + | ==Procedures== |
- | + | ===Cell lysis=== | |
- | # Pellet cells by centrifugation at | + | # Pellet cells by centrifugation at 3600 rpm for 10 minutes. |
# Pour off supernatant. | # Pour off supernatant. | ||
- | # Add 0. | + | # Add 0.5 ml of cell suspension solution, gently pipet up and down to resuspend and transfer to 1.5 ml eppendorf tube. |
- | # Add | + | # Add 25 μl of lysozyme and invert 25 times. |
# Incubate for 30 minutes at 37°C inverting occasionally. | # Incubate for 30 minutes at 37°C inverting occasionally. | ||
- | # Centrifuge at | + | # Centrifuge at 13000 rpm for 10 minutes to pellet the cells then remove the supernatant. |
- | # Add 0. | + | # Add 0.5 ml of cell lysis solution to the cell pellet and gently pipet up and down to lyse the cells. |
# Heat sample for 30 minutes mix every 5-10 minutes. | # Heat sample for 30 minutes mix every 5-10 minutes. | ||
- | + | ===RNase treatment=== | |
- | # Add 3 | + | # Add 3 μl 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. | # Cool samples on ice. | ||
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# Centrifuge at 13000 rpm for 30 seconds or until the precipitated proteins form a tight pellet. | # Centrifuge at 13000 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.) | # 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. | + | # Add 0.5 ml isopropanol to each tube. |
# Mix by inverting gently for 50 times. | # Mix by inverting gently for 50 times. | ||
# Centrifuge at 13000 rpm for 1 minute. The DNA should be visible as a small white pellet. | # Centrifuge at 13000 rpm for 1 minute. The DNA should be visible as a small white pellet. | ||
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# Drain the tubes on clean absorbent paper. Allow to air dry for 10-15 minutes. | # 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. | # 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. | # 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. | # For storage, centrifuge briefly and store at -20°C. | ||
+ | |||
+ | {{Team:Newcastle/footer}} |
Revision as of 15:19, 28 July 2010
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Contents |
DNA extraction
Materials Required
- Cells grown from yesterday
- Centrifuge
- pipette
- lysozyme
- Cell lysis solution
- RNase solution
- protein precipitation solution
- ice
- isopropanol
- ethanol
Procedures
Cell lysis
- Pellet cells by centrifugation at 3600 rpm for 10 minutes.
- Pour off supernatant.
- Add 0.5 ml of cell suspension solution, gently pipet up and down to resuspend and transfer to 1.5 ml eppendorf tube.
- Add 25 μl of lysozyme and invert 25 times.
- Incubate for 30 minutes at 37°C inverting occasionally.
- Centrifuge at 13000 rpm for 10 minutes to pellet the cells then remove the supernatant.
- Add 0.5 ml of cell lysis solution to the cell pellet and gently pipet up and down to lyse the cells.
- Heat sample for 30 minutes mix every 5-10 minutes.
RNase treatment
- Add 3 μl 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 13000 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 0.5 ml isopropanol to each tube.
- Mix by inverting gently for 50 times.
- Centrifuge at 13000 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 13000 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.