DNA extraction using Chelex 100: Difference between revisions
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* Fill half a tube (500ul) with chelex 100 (try and get 50% beads and 50% clear liquid) | ==New Protocol== | ||
We tend to use the protocol made available online here: [http://biology.clc.uc.edu/fankhauser/Labs/Genetics/Buccal_DNA_isolation/buccal_dna_images_index.html DNA extraction from buccal cells]. | |||
In summary: | |||
# Mix a pinch of '''salt''' with about 100mL '''water''' (about a cappucino cup) | |||
# Take a sip and '''swish''' it around in your mouth; chewing on your cheeks can improve results | |||
# Expel 10mL of the result into a 15mL propylene tube, do this over the sink for better hygiene | |||
# Place the tube into the '''centrifuge''' with appropriate counterweight and spin for 10min | |||
#* At this point you may want to start setting up the hob plate to get some water boiling | |||
# You should see a pellet at the bottom of the tube. These are your cells: '''decant the supernatant''' (liquid on top) entirely | |||
# Use a clean pasteur pipette to transfer 0.5mL (500μL) of 5% '''chelex 100''' into the tube (try and get 50% beads and 50% clear liquid) | |||
# Pipette up and down to '''mix''' until there are no visible cell lumps left | |||
# Close the tube and place it in a beaker of '''boiling''' water for 10min to lyse the cells | |||
#* Try to avoid the tube touching the bottom of the beaker (because it may be hotter than 100°C and damage DNA) | |||
# Use a pasteur pipette (can be the same as above) to transfer the contents into a '''1.5mL Eppendorf tube''' | |||
#* It's no problem if you can't get the beads, just make sure you get as much of the liquid as possible | |||
# Spin in the small '''centrifuge''' with appropriate counterweight at max speed for 30-60s | |||
# Use the micropipettor to '''transfer''' 200μL of the '''supernatant''' to a new tube. Make sure you take nothing from the pellet | |||
#* The pellet at the bottom contains protein and lipids, which you want to avoid. The DNA is in the supernatant | |||
# Store at -20°C | |||
==Old Protocol== | |||
* Fill half a tube (500ul) with 5% chelex 100 (try and get 50% beads and 50% clear liquid) | |||
* Scrape off some cheek cells into the end of a pipette tip, add this and some saliva to the tube and mix | * Scrape off some cheek cells into the end of a pipette tip, add this and some saliva to the tube and mix | ||
* Cover with a drop of paraffin Incubate at 56°C for 30 mins, then at 94°C for 10 mins | * Cover with a drop of paraffin Incubate at 56°C for 30 mins, then at 94°C for 10 mins | ||
* Centrifuge briefly and take supernatant | * Centrifuge briefly and take supernatant | ||
* Store at -20°C | * Store at -20°C | ||
[[Category:Biohacking]] | |||
[[Category:Guides]] | |||
Latest revision as of 20:21, 26 January 2015
New Protocol
We tend to use the protocol made available online here: DNA extraction from buccal cells.
In summary:
- Mix a pinch of salt with about 100mL water (about a cappucino cup)
- Take a sip and swish it around in your mouth; chewing on your cheeks can improve results
- Expel 10mL of the result into a 15mL propylene tube, do this over the sink for better hygiene
- Place the tube into the centrifuge with appropriate counterweight and spin for 10min
- At this point you may want to start setting up the hob plate to get some water boiling
- You should see a pellet at the bottom of the tube. These are your cells: decant the supernatant (liquid on top) entirely
- Use a clean pasteur pipette to transfer 0.5mL (500μL) of 5% chelex 100 into the tube (try and get 50% beads and 50% clear liquid)
- Pipette up and down to mix until there are no visible cell lumps left
- Close the tube and place it in a beaker of boiling water for 10min to lyse the cells
- Try to avoid the tube touching the bottom of the beaker (because it may be hotter than 100°C and damage DNA)
- Use a pasteur pipette (can be the same as above) to transfer the contents into a 1.5mL Eppendorf tube
- It's no problem if you can't get the beads, just make sure you get as much of the liquid as possible
- Spin in the small centrifuge with appropriate counterweight at max speed for 30-60s
- Use the micropipettor to transfer 200μL of the supernatant to a new tube. Make sure you take nothing from the pellet
- The pellet at the bottom contains protein and lipids, which you want to avoid. The DNA is in the supernatant
- Store at -20°C
Old Protocol
- Fill half a tube (500ul) with 5% chelex 100 (try and get 50% beads and 50% clear liquid)
- Scrape off some cheek cells into the end of a pipette tip, add this and some saliva to the tube and mix
- Cover with a drop of paraffin Incubate at 56°C for 30 mins, then at 94°C for 10 mins
- Centrifuge briefly and take supernatant
- Store at -20°C
