The most important: 2 bottles of beer (33cl). In our first prototype, we used a Chimay red. Using a non-filtered beer should give more DNA for sequencing.
Magnetic beads (SPRI beads like AMPure Beads XP or similar)
It is possible to prepare cheap homemade SPRI beads to purify DNA by following instructions
P10, P20, P200, P1000, P5000 pipette
Pre-cooled the centrifuge so that it starts at 4°C
This part is optional. But we hypothesize that keeping the beer at the preferred drinking temperature improves the sequencing results [proof is needed].
Open the beer few hours before to get most of the CO2 out
Shake the beer bottle (a bit)
Often the yeast’s settle down to the bottom.
Transfer into an 1000ml Erlenmeyer flask
Shake it carefully the Erlenmeyer flask to remove the CO2. A foam will form, whose the size depends on the beer, its temperature and for how long it was open.
Transfer the beer (not the foam) into 50ml Falcon tubes using a large pipette
Put the tubes (with the lids closed) in the centrifuge for 10min at full-speed
Be careful that the centrifuge is correctly balanced: put same number of tubes on each opposite side.
This step separates the liquid phase and the solid phase (which contains yeast among other things):
Discard carefully after centrifugation the supernatant either by pipetting or by pouring the supernatant into the sink
Be sure that the pellet remains in the Falcon
Add 1mL of Tris-buffer pH 7.4 to the Falcon with pellet with a pipette
Mix it to the pellet (the solid part on the bottom) and resuspend it the content by pipetting up and down a few times
The pellet will go into solution and disappear. Afterwards, no solid phase should be visible and the solution should turn into a brownish color.
Transfer the solution into 1.5ml Eppendorf tubes
We now want to get the DNA out the yeast. The DNA is well protected by the membrane of the nucleus and the membrane of the cell. We need to break the membrane of the yeast and then the menbrane of the nucleus.
A yeast cell - Frankie Robertson, CC ASA, Wikimedia
The freezing and boiling destroys both membranes and help to release the DNA.
Now we want to extract DNA. The liquid contains also buffer and cell garbage.
To extract the DNA, we will make the DNA binding to some beads and then use a magnet to capture the beads with the DNA. Then, we wash and repeat pretty much the same step: that time, beads will attach to the tube and the liquid will contain our DNA.
Centrifuge the sample at highspeed for 5 min
A pellet will appear at the bottom of the tube
Transfer the supernatant to a fresh tube (only the supernatant and not the pellet)
Take a P1000 pipette and check if the volume of your solution is 200 µl
We need a certain ratio of beads given the DNA we have
Vortex the SPRI beads very carefully
It has to be a smeary brownish solution (to be nerdy: actually, it’s more a dispersion than a solution).
Add the needed quantity of beads (1.7 x of the volume in your tube, i.e. 340 µl beads) to the sample
The tube has now a volume of approximatively 540 µl.
Place/fix your tube to either a magnet or a magnetic rack to attach the beads for 1 min
The liquid should get clear as the magnetic beads will attach at the wall of the tube towards the magnet.
Remove carefully the clear liquid from the tubes using pipette without touching/disturbing the brown beads
To do so, the tube has to be at the magnet/in the magnetic rack.
Remove carefully the liquid
It might be hard to aspirate the very last drop of ethanol with your pipette. You can place the tube into the centrifuge to collect everything at the bottom and place the tube immediately at the magnet. Then, remove the last drops using a pipette.
Dry for 2 min with open lid
During this time, ethanol residues will evaporate.
Remove the tube from the magnet and mix properly (vortex)
The water is more attractive for the DNA than the beads, so it will go into solution.
Put the tubes back to the magnet
Be aware! The clear solution contains now the DNA.
The liquid in the tube now contains the DNA
The liquid still contains some cell garbage. To improve the purity we will use some columns with resine on which the DNA will bind.
Umprove the purity
Pipette the samples into the column
The column contains a silica based material and the DNA in our yellowish solution will bind to this material. All other things will be washed away by centrifugation.
Centrifuge for 1 min at full speed
The DNA binds to the column and stays there.
Centrifuge for 1 min to elude the DNA from the column
The liquid in the tube now contains the DNA.
Check the purity with a spectrophotometer
Spectrowhat?! A spectrophotometer sends light with different wavelengths through our DNA-sample. DNA absorbs light at a defined wavelength (maximum absorption at 260 nm wavelength). This can be used to quantify the amount of DNA. Concerning Nanopore sequencing: The more, the better! Furthermore, we can estimate the purity of our DNA. EtOH for example has an absorption maximum at 230 nm. At 230 nm there should be no peak in the sprectrum (or at least much smaller than the DNA-peak at nm=260).
Well done! Now you have successfully extracted beer DNA! Go on and sequence your extracted DNA or visit the next pub…