15
edits
DNA extraction using Chelex 100: Difference between revisions
From London Hackspace Wiki
DNA extraction using Chelex 100 (view source)
Revision as of 18:26, 25 February 2013
, 25 February 2013new protocol
Mycoplasma (talk | contribs) No edit summary |
(new protocol) |
||
| Line 1: | Line 1: | ||
==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) | * 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 | ||
| Line 4: | Line 26: | ||
* Centrifuge briefly and take supernatant | * Centrifuge briefly and take supernatant | ||
* Store at -20°C | * Store at -20°C | ||
[[Category:Biohacking]] | |||
