Extraction protokoll

_protocols/beer-dna-sequencing.md

@@ -4,19 +4,46 @@ title: Beer DNA sequencing
 image: /images/protocols/beer-dna-sequencing.jpg
 ---
 
-## Requirements
+## Requirements Software setup
+
+### MinKNOW
+
+MinKNOW is the program needed to connect your computer with MinION. It has a graphic user interface for configuring and running the sequencing. Furthermore, it has integrated base-caller software to convert the raw nanopore signals to the strings of nucleotides, in fastq format.
+
+### Installation:
+
+- Please search for "MinION Software" in the Nanopore downloads web page https://community.nanoporetech.com/downloads.
+- Select your host operating system and follow the link to the installation instructions.
+- Since the supported systems and the installation procedure is constantly updated, please always refer to the community website for details. (You need to have an account to access the community website)
+
+### Setting up the software:
+
+#### Requirements:
+- You have to have a account at nanopor MinION to start the software
+
+#### Do: 
+- Attech the MinION divice to your computer (connected to through the USB 3.0 port)
+- Open the divice; remove the addapter and add a flowcell instead 
+- To start the software you either find the "MinION Software" botton directly or you can open it via the terminal using the path "/opt/ui/MinKNOW"
+- After starting the software, first select the flow cell type. You should find the type disctiption on the flowcell package
+- Click on the botton 'Check flow cell' and 'start test'
+- On the right side under massanges you will see when the check is compleated and how may nanopores are avalabel for your sequencing run.
+
+
+
+## Requirements DNA
 
 The most important: **7.5µl DNA** extracted as described in the [DNA extraction protocol]({% link _protocols/beer-dna-extraction.md %})
 
 
 ### Before starting Prepare DNA
 
 - DNA ~ 400 ng should be in 7.5 µl nuclease-free water
-- DNA should be mixed by flicking the tube and spin down briefly in a microfuge or (micro-centrifuge)
+- DNA should be mixed by flicking the tube and spin down briefly in a centrifuge
 - All kit reagents should be:
 1. Thaw to room temperature
 2. Briefly spin down
-3. Mix well by pipetting
+3. Mix well by pipetting (up and down)
 - Once thawed, keep all the kit components on ice
 - Always repeat the mixing step before using each reagent
 
@@ -28,18 +55,19 @@ The most important: **7.5µl DNA** extracted as described in the [DNA extraction
 - Fragmentation Mix (FRA)
 - Rapid Adapter (RAP)
 - Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
-- 0.2 ml thin-walled PCR tubes
+- 0.2 ml PCR tubes
 - Ice
 
 ##### Needed material
 - Thermal cycler at 30° C and 80° C
 - P2 pipette and tips
 - P10 pipette and tips
+- centrifuge
 
 
 #### Do
 
-1. Prepare tubes with 7.5 µl extracted DNA and 2.5 µl FRA
+1. Prepare a tube with 7.5 µl extracted DNA and 2.5 µl FRA
 2. Mix gently by flicking the tube, and spin down
 3. Put the tubes into a thermocycler: 1 min at 30℃, 1 min at 80℃
 4. Keep on ice until next step
@@ -57,8 +85,8 @@ The most important: **7.5µl DNA** extracted as described in the [DNA extraction
 - Flush Buffer (FB)
 - Loading Beads (LB)
 - Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
-- 1.5 ml Eppendorf DNA LoBind tubes
-- Flongle device - flow cell and adapter
+- 1.5 ml Eppendorf DNA 
+- MinIon diveice including Flow cell
 - Ice
 
 ##### Needed material
@@ -67,21 +95,24 @@ The most important: **7.5µl DNA** extracted as described in the [DNA extraction
 - P1000 pipette and tips
 - P20 pipette and tips
 - P10 pipette and tips
+- Vortex
+- centrifuge
 
 
-#### Prepare loading port
 
+#### Prepare substance of the priming mix
 1. Mix the Sequencing Buffer (SQB) and Flush Buffer (FB) tubes by vortexing, spin down and return to ice
 2. Spin down the Flush Tether (FLT) tube, mix by pipetting, and return to ice
-3. Open priming port to check for small bubbles
-4. If there are bubbles remove them by taking some liquid from the port
-5. To remove use the P1000 pipette set to 200 µl
-6. Remove the liquid by turning the weel but only until 220-230 µl
-7. Removing more than 30 µl will damage the pores in the array because they need to be covered by the buffer at all times
+
+#### Prepare loading port
+1. Open priming port to check for small bubbles
+2. To remove bubbles take some liquid from the port by use the P1000 pipette set to 200 µl
+3. Remove the liquid by turning the weel but only until 220-230 µl
+4. Note: removing more than 30 µl will damage the pores in the array because they need to be covered by the buffer at all times
 
 #### Prepare flowcell priming mix
 
-1. Add 30 µl of Flush Tether (FLT) directly to the Flush Buffer (FB) Eppi tube and mix by pipetting up and down
+1. Add 30 µl of Flush Tether (FLT) directly to the Flush Buffer (FB) Eppi tube and mix by pipetting 
 2. Load 800 μl of the priming mix into the flow cell via the priming port, avoiding introduction of air bubbles
 3. Wait for 5 minutes
 
@@ -103,23 +134,7 @@ The most important: **7.5µl DNA** extracted as described in the [DNA extraction
 
 ## Protocol: Software setup
 
-### MinKNOW
-
-MinKNOW is the program needed to connect your computer with MinION. It has a graphic user interface for configuring and running the sequencing. Furthermore, it has integrated base-caller software to convert the raw nanopore signals to the strings of nucleotides, in fastq format.
-
-#### Installation:
-
-- Please search for "MinION Software" in the Nanopore downloads web page https://community.nanoporetech.com/downloads.
-- Select your host operating system and follow the link to the installation instructions.
-- Since the supported systems and the installation procedure is constantly updated, please always refer to the community website for details.
-
-#### Starting the software:
-
-Requirements:
-- You have to have a account at nanopor MinION to start the software
-- The divice need to be attached to the computer to start the software
 
-Either you find the "MinION Software" botton directly or you open it via the terminal using the path "/opt/ui/MinKNOW" 
 
 #### Launching MinKNOW and running the sequencing
 

_protocols/new-dna-extraction.md

@@ -0,0 +1,147 @@
+---
+layout: default
+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: 
+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)
+
+
+### 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.
+
+	![](/images/protocols/beer-dna-extraction/erlenmeyer_with_beer.svg){: width="35%"}
+
+4. Transfer the beer (not the foam) into 50 ml tubes
+  ![](/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)
+
+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%"}
+
+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).
+
+    ![](/images/protocols/beer-dna-extraction/buffer_collection.svg){: width="25%"}
+
+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%"}
+
+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
+
+
+
+### 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)*
+
+- Suspend cells:
+
+	1. Add 640µl of the Y-PER Reagent.
+
+		[Y-PER](https://www.thermofisher.com/order/catalog/product/78991#/78991) is a detergent optimized for yeast cell lysis. 
+
+		The amount of Y-PER reagent is calculated by taking the ratio of 8μL(reagent)/1mg pellet
+
+		For simplification we assume all pellets correspond to 80 mg and added 640µl Y-PER
+
+	2. Mix by pipetting up and down until the mixture is homogenous
+
+- Incubate at 65°C for 10 - 30 minutes.
+- Centrifuge at 13,000 rcf for 5 minutes 
+- Discard supernatant
+
+
+### STEP 3: Break-down the yeast cell wall – second round
+
+- Add 400μL of DNA Releasing Reagent A
+- Add 400μL of DNA Releasing Reagent B
+
+	A protein Removal Reagent can be protease to digest proteins or a salt solution to precipitate protein (salting-out).
+
+- Mix by pipetting up and down until the mixture is homogenous
+- Incubate at 65°C for 10 - 30 minutes. 
+
+
+### 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 
+
+
+### 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: liquit above you), being careful not to discard any of the pellet, which is clear and hard to see.
+
+	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. 
+
+
+### 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)
+
+### STEP 7: Resuspend the DNA
+
+- 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...
+