Updated Extraction Protocol

SaimMomin12 · SaimMomin12 · commit a5f8790e2a19 · 2024-07-15T15:29:27.000+02:00
diff --git a/_protocols/beer-dna-extraction.md b/_protocols/beer-dna-extraction.md
@@ -1,214 +1,156 @@
 ---
 layout: default
-title: Beer DNA extraction
+title: new Beer DNA extraction
 image: /images/protocols/beer-dna-extraction.jpg
 ---
 
 ## Requirements
 
 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.
 
-Needed consumables
-- 10x 50ml Falcon tubes
-- 4x 1.5ml Eppendorf tubes
-- 4ml Tris-buffer pH7.4 1M
-- Pipette tips
-- Ethanol bath or dry ice
-- 2ml Lysis buffer: 2% Triton X-100, 1% SDS, 10mM NaCl, 10mM Tris-HCl, 1mM EDTA
-- 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](https://tinyurl.com/yyk9dz8c)
-
-- 70% Ethanol solution
-- DNA binding column, i.e. [QIAquick PCR Purification Kit](https://tinyurl.com/y29rg4my) or MinElute Reaction Kit 50 from Qiagen
-- 750µl Buffer PE
-- Nuclease free water (for solving the DNA pellet)
-
-Needed material
-- 1x 1000ml Erlenmeyer
-- 1x serological Transfer Pipette
-- P10, P20, P200, P1000, P5000 pipette
-
-  ![](/images/protocols/pipette.svg){: width="20%"}
-
-- (Thermo-regulated) Centrifuge
-- Water bath at 100°C
-- Tape
-- Vortex
-- Magnetic rack or strong magnet
-- Spectrophotometer (Nanodrop)
-
-## Beer DNA extraction
-
-1. 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].
-
-2. Open the beer few hours before to get most of the CO2 out
-
-### Extract the yeast from the beer
-
+Needed consumables: 
+1. Yeast DNA Extraction Kit Thermo Fischer 
+2. TrisHCl-buffer (1M, pH 7.4)
+	-1.0mL per sample + 50ml per sample (washing)
+3. 70% EtOH (for molecular biology)
+	- 1.5mL per sample
+4. Isopropanol (for molecular biology)
+	- 600µL per sample
+5. Sterile water (for molecular biology)
+	- 50µL per sample
+6. Sterile eppis (1.5mL)
+	- 2 per sample
+7. (Falcon) tubes (according to beer volume)
+8. Ice
+
+## Material
+- 1000 ml Erlenmeyer flask
+- Glass pipette (25mL)
+- P1000 pipette and tips
+- P200 pipette and tips
+- P100 pipette and tips
+- Centrifuge (loadable with Falcon tubes)
+- Centrifuge
+- Thermo block
+
+### Before starting
+- Prewarm DNA Releasing Reagent A and B at 37°C for 5 min 
+- Pre-cooled the centrifuge and tubes so that it starts at 4°C, because we hypothesize that keeping the beer at the preferred drinking temperature improves the sequencing results [proof is needed].
+- Switch on thermo block at 65°C
+
+### STEP 1: Harvest the yeast from the beer
 1. Shake the beer bottle (a bit)
+2. Transfer into a 1000 ml Erlenmeyer flask (the big glass that looks like a triangle)
+3. You should carefully shake the Erlenmeyer to remove most of the CO2. A foam will form, whose size depends on the beer, its temperature and for how long it has been open.
 
-    Often the yeast's settle down to the bottom.
-
-2. 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.
-
-    ![](/images/protocols/beer-dna-extraction/erlenmeyer_with_beer.svg){: width="35%"}
+	![](/images/protocols/beer-dna-extraction/erlenmeyer_with_beer.svg){: width="35%"}
 
-3. Transfer the beer (not the foam) into 50ml Falcon tubes using a large pipette
+4. Transfer the beer (not the foam) into 50 ml tubes
+  ![](/images/protocols/beer-dna-extraction/falcon_with_beer.svg){: width="75%"}
 
-    ![](/images/protocols/beer-dna-extraction/falcon_with_beer.svg){: width="75%"}
-   
     - Make sure each tube gets the same quantity (to balance the centrifuge for the next step)
     - Put a lid on each tube but don't close them until the next step (CO2 needs to be evacuated)
-   
-4. 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.
-   
+5. Centrifuge at 4000 rpm and 4°C for 10 min
+    Be careful that the centrifuge is correctly balanced: for each tube put one on the opposite side.
+
     This step separates the liquid phase and the solid phase (which contains yeast among other things): 
 
     ![](/images/protocols/beer-dna-extraction/after_centrifuge_1.svg){: width="25%"}
 
-5. 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
+6. Discard carefully the supernatant either by pouring the liquid phase. But anyway, be sure that the pellet remains in the tube.
+7. Transfer 1 ml of TrisHCl-buffer (1M, pH 7.4) to the tube. 
+  - the buffer helps to separate the yeast cells form the rest of the beer (washing).
 
-6. Add 1mL of Tris-buffer pH 7.4 to the Falcon with pellet with a pipette
+    ![](/images/protocols/beer-dna-extraction/buffer_collection.svg){: width="25%"}
 
-   ![](/images/protocols/beer-dna-extraction/buffer_collection.svg){: width="25%"}
-
-7. Mix it to the pellet (the solid part on the bottom) and resuspend it the content by pipetting up and down a few times
+8. Mix by pipetting to resolve the pellet (Aspirate and pull out the liquid a couple of time with the pipette. You will see that the pellet will go into solution and disappear.) Afterwards, no solid phase should be visible and the solution should turn into a brownish color.
 
    ![](/images/protocols/beer-dna-extraction/suspend_pellet.svg){: width="85%"}
 
-   The pellet will go into solution and disappear. Afterwards, no solid phase should be visible and the solution should turn into a brownish color.
-
-6. Transfer the solution into 1.5ml Eppendorf tubes
-
+9. Fill up to 20ml whith the TrisHCl-buffer 
+10. Centrifuge 4000 rpm 10 min 4°C
+11. Discard supernatant
+12. Resuspend the cells (with ca. 1ml 1M TrisHCl buffer pH 7.4) 
+13. Transfer the solution into a 1.5 ml Eppendorf tube (eppi).
    ![](/images/protocols/beer-dna-extraction/transfer_to_eppendorf.svg){: width="20%"}
+14. Centrifuge 8000 rcf 5 min 4°C
+15. Discard supernatant
+16. Weigh each of your eppy pellets (use empty 1.5ml eppi as tara): weights of one pellet between 30mg and 60mg
+17. Now we like to come to approx. 70-90mg pellet per eppi: 
+	- Resolve the pellets by adding 500 μL and pipet up and down 
+	- Eventually combine or split the solution of eppis to achieve ca. 70-90 mg pellet per eppi
+	- Centrifuge 8000 rcf 5 min 4°C
+	- Discard supernatant
 
-8. Label the tubes
-9. Put the tubes into the centrifuge one more time for about 5 min at full-speed
-10. Discard the supernatant by pipetting
 
-### Break the cell walls of the yeasts and nucleus
 
-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.
+### STEP 2: Break-down the yeast cell wall – first round
+Now, we want to get the DNA out the yeast. The DNA is well protected by the cell wall and the membrane of the nucleus. We need to break the membrane of the yeast and then the menbrane of the nucleus.
 
 ![](/images/protocols/beer-dna-extraction/yeast_cell.svg){: width="50%"}
 
 *A yeast cell - Frankie Robertson, CC ASA, [Wikimedia](https://en.wikipedia.org/wiki/File:Yeast_cell_english.svg)*
 
-1. Prepare a cold mix (-20°C) with 3 volume of ice and 1 volume of salt
-1. Add 200µl of lysis buffer into each tube
-2. Mix carefully by pipetting up and down (at least 10 times) to resolve the pellet
-3. Repeat 2 times
-    1. Put the tubes into the cold mix (-20°C) for 2 min
-    2. Put the tubes in boiling water (100°C) for 2 min
-    3. Vortex the tubes for 30s
-
-The freezing and boiling destroys both membranes and help to release the DNA.
-  
-### Extract the DNA from the cell
-
-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.
-
-1. Centrifuge the sample at highspeed for 5 min
-
-    A pellet will appear at the bottom of the tube
-
-2. Transfer the supernatant to a fresh tube (only the supernatant and not the pellet)
-
-7. Place/fix your tube to the magnet
-2. Bind DNA to the magnetic beads
-    1. 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
-
-    2. 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).
-
-    3. 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.
-
-    4. Vortex the tube and incubate for 5 min
-    3. Place/fix your tube to either a magnet or a magnetic rack to attach the beads for 1 min
-
-       ![](/images/protocols/beer-dna-extraction/beads.svg){: width="25%"}
-
-       The liquid should get clear as the magnetic beads will attach at the wall of the tube towards the magnet.
-
-3. Washing
-    1. 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.
-
-    2. Add 100µl of 70% Ethanol solution into each tube (still on the magnet)
-    3. 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.
-
-    4. Put the tubes back to the magnet
-    5. Open the lid of the tube
-    6. Dry for 2 min with open lid
+- Suspend cells:
 
-       During this time, ethanol residues will evaporate.
+	1. Add 640µl of the Y-PER Reagent.
 
-    7. Add 100µl of nuclease free water
-    8. Remove the tube from the magnet and mix properly (vortex)
+		[Y-PER](https://www.thermofisher.com/order/catalog/product/78991#/78991) is a detergent optimized for yeast cell lysis. 
 
-        The water is more attractive for the DNA than the beads, so it will go into solution. 
+		The amount of Y-PER reagent is calculated by taking the ratio of 8μL(reagent)/1mg pellet
 
-    9. Incubate for 2 min
-    10. Put the tubes back to the magnet
+		For simplification we assume all pellets correspond to 80 mg and added 640µl Y-PER
 
-        Be aware! The clear solution contains now the DNA.
+	2. Mix by pipetting up and down until the mixture is homogenous
 
-    11. Transfer the clear liquid to a fresh tube (1.5ml)
+- Incubate at 65°C for 10 - 30 minutes.
+- Centrifuge at 13,000 rcf for 5 minutes 
+- Discard supernatant
 
-    The liquid in the tube now contains the DNA
 
+### STEP 3: Break-down the yeast cell wall – second round
 
-    The liquid still contains some cell garbage. To improve the purity we will use some columns with resine on which the DNA will bind.
+- Add 400μL of DNA Releasing Reagent A
+- Add 400μL of DNA Releasing Reagent B
 
-4. Umprove the purity
+	A protein Removal Reagent can be protease to digest proteins or a salt solution to precipitate protein (salting-out).
 
-    1. Add 500µl of PB-buffer (from the column kit) to the sample (the yellowish solution) and vortex
-    2. Place a column into a collection tube
-    3. Pipette the samples into the column
+- Mix by pipetting up and down until the mixture is homogenous
+- Incubate at 65°C for 10 - 30 minutes. 
 
-        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.
 
-        ![](/images/protocols/beer-dna-extraction/column.svg){: width="25%"}
+### STEP 4: Stop protein activity in the solution
+- Add 200μL of Protein Removal Reagent to mixture
+- Invert eppy several times (>20x)
+- Centrifuge at least 13,000 rcf for 5 minutes 
+- Transfer supernatant (only 900µl!!!!!) to a new 1.5mL eppy
+  - try to not touch the pellet with the pipet tip 
 
-    4. Centrifuge for 1 min at full speed
 
-        The DNA binds to the column and stays there.
+### STEP 5: Separate the DNA from other molecules
+- Add 600μL isopropyl alcohol to fill tube
+- Invert eppy several times gently (>20x)
+- Separate DNA by centrifuging the mixture at 13,000 rcf for 10 minutes. 
+- The DNA should be at the bottom of the eppy (pellet)
+- Remove supernatant (here: liquid above you pellet), being careful not to discard any of the pellet, which is clear and hard to see.
 
-    5. Add 750µl Buffer PE to the column (which is nothing else than 70% ethanol)
-    7. Centrifuge for 1 min at full speed
-    8. Remove the liquid from the collection tube and put the column back
-    9. Centrifuge for 1 min to dry the column
-    10. Transfer the column content out into a new Falcon tube
-    11. Discard the collection tube
-    12. Add 11µl water to the column and wait for 1 min
-    13.	Centrifuge for 1 min to elude the DNA from the column
+	DNA is negatively charged, therefore hydrophilic (dissolves in water). The carbon chain of alcohol is hydrophobic, so the DNA is less soluble and precipitates. Isopropanol and ethanol are alcohol. Isopropanol has a longer carbon chain than ethanol (therefore more hydrophobic) and thus precipitates DNA stronger than ethanol.
+The problem is that isopropanol also precipitates salts. To remove salts, we wash the DNA pellet with ethanol in STEP 6. 
 
-            The liquid in the tube now contains the DNA.
 
-5. Check the purity with a spectrophotometer
+### STEP 6: Wash the DNA to remove unwanted substances
+- Add 1.5mL of 70% ethanol to the pellet
+- invert eppy several times (>20x)
+- Centrifuge at 13,000 rcf for 1 minute to wash off any residual salts or cellular debris clinging to the DNA or tube. 
+- Invert the eppy carfully but in one go to remove the liquide, without damageing the pellet
+- to dry any residual ethanol before proceeding to Step 7 place the eppy up side down on a tissue. (took approx. 30-45min)
 
-    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).
+### STEP 7: Resuspend the DNA
 
-6. Freeze the DNA until library preparation
+- add 50μL sterile water to each eppy
+- Flick the bottom of the tube carefully, or pipette solution up and down
+- Wash the sides of the tubes until all the genomic DNA is in solution (should take 5 min)
+- Freeze the DNA until library preparation or start directly!
 
-Well done! Now you have successfully extracted beer DNA! [Go on and sequence your extracted DNA]({% link _protocols/beer-dna-sequencing.md %}) or visit the next pub!
+Well done! Now you have successfully extracted beer DNA! [Go on and sequence your extracted DNA]({% link _protocols/beer-dna-sequencing.md %}) or visit the next pub!
