From ed532c2c65a4f2df6abc9e1c4c2a0a2164456b5d Mon Sep 17 00:00:00 2001 From: muellert Date: Thu, 7 Nov 2019 14:06:01 +0100 Subject: [PATCH 1/8] added discussed changes of the last sequencing round to the protokoll --- _protocols/beer-dna-sequencing.md | 75 ++++++++++++++++++------------- 1 file changed, 45 insertions(+), 30 deletions(-) diff --git a/_protocols/beer-dna-sequencing.md b/_protocols/beer-dna-sequencing.md index ed2c6d3..15909c4 100644 --- a/_protocols/beer-dna-sequencing.md +++ b/_protocols/beer-dna-sequencing.md @@ -4,7 +4,34 @@ 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 %}) @@ -12,11 +39,11 @@ The most important: **7.5µl DNA** extracted as described in the [DNA extraction ### 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 From 37ea45711ffa742612e0e0c07204243822381b12 Mon Sep 17 00:00:00 2001 From: muellert Date: Sun, 24 Nov 2019 10:46:21 +0100 Subject: [PATCH 2/8] added the new extraction protokoll --- _protocols/new-dna-extraction.md | 90 ++++++++++++++++++++++++++++++++ 1 file changed, 90 insertions(+) create mode 100644 _protocols/new-dna-extraction.md diff --git a/_protocols/new-dna-extraction.md b/_protocols/new-dna-extraction.md new file mode 100644 index 0000000..139ad96 --- /dev/null +++ b/_protocols/new-dna-extraction.md @@ -0,0 +1,90 @@ +--- +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. TrisHCl-buffer (1M, pH 7.4) + -1.0mL per sample + 50ml per sample (washing) +2. 70% EtOH (for molecular biology) + - 1.5mL per sample +3. Isopropanol (for molecular biology) + - 600µL per sample +4. Sterile water (for molecular biology) + - 50µL per sample +5. Sterile eppis (1.5mL) + - 2 per sample +6. Falcon tubes (according to beer volume) +7. Cool down centrifuge for falcon tubes +8. Switch on thermo block at 65°C + + + +### Before starting +Prewarm DNA Releasing Reagent A and B at 37°C for 5 min + +### 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) + - You should carefully shake it to remove most of the CO2 +3. Transfer the beer (not the foam) into 50 ml Falcon tubes +4. Centrifuge 4000 rpm 10 min 4°C +5. Discard carefully the supernatant either by pouring the liquid phase. But anyway, be sure that the pellet remains in the Falcon. +6. Transfer 1 ml of TrisHCl-buffer (1M, pH 7.4) to the Falcon. 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 turned into a brownish color. +7. Fill up to 20ml +8. Centrifuge 4000 rpm 10 min 4°C +9. Discard supernatant +10. Resuspend the cells (with ca. 1ml 1M TrisHCl buffer pH 7.4) +11. Transfer the solution into a 1.5 ml Eppendorf tube. +12. Weigth pellets (fresh empty 1.5ml eppi as tara): 6 eppis, weights between 30mg and 60mg +13. Pool pellets from two eppis to achieve ca. 70-90mg pellet per eppi (final # of eppis: 3) + + + +### STEP 2: Break-down the yeast cell wall – first round +Here: incubation times can be prolonged for difficult to lyse organisms +- Suspend cells in an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. + - We assumed all pellets correspond to 80 mg and added 640µl Y-PER +- Mix by pipetting up and down until the mixture is homogenous +- Incubate at 65°C for 10 minutes. (can be extended – SHOULD BE EXTENDED??) + + +### STEP 3: Break-down the yeast cell wall – second round +Here: incubation times can be prolonged for difficult to lyse organisms +- Centrifuge at 13,000 × g for 5 minutes, +- Discard supernatant, +- Add 400μL of DNA Releasing Reagent A, +- Add 400μL of DNA Releasing Reagent B +- Mix by pipetting up and down until the mixture is homogenous +- Incubate at 65°C for 10 minutes. (can be extended – SHOULD BE EXTENDED??) + + +### STEP 4: Stop protein activity in the solution +- Add 200μL of Protein Removal Reagent to mixture +- Invert several times (>20x). +- Centrifuge at least 13,000 × g for 5 minutes +- Transfer supernatant (only 900µl!!!!!) to a new 1.5mL centrifuge tube. + + +### STEP 5: Precipitate (find a non-scientific word!) the DNA +- Add 600μL isopropyl alcohol to fill tube. +- Mix gently by inversion. (>20x). +- Precipitate genomic DNA by centrifuging the mixture at 13,000 × g for 10 minutes. + + +### STEP 6: Wash the DNA to remove unwanted substances +- Remove supernatant, being careful not to discard any of the pellet, which is clear and hard to see. +- Add 1.5mL of 70% ethanol to the pellet, invert several times (>20x). +- Centrifuge at 13,000 × g for 1 minute to wash off any residual salts or cellular debris clinging to the DNA or tube. +- Invert the tube to dry any residual ethanol before proceeding to Step 7. (took ca. 30-45min) + +### STEP 7: Resuspend the DNA + +- Resuspend in 50μL sterile water. Pellet should solubilize completely within 5 minutes (not really). 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. + + From 8623daa90bcfd21fd4dfb5041ab61fd58cd78657 Mon Sep 17 00:00:00 2001 From: muellert Date: Sun, 23 Feb 2020 18:51:12 +0100 Subject: [PATCH 3/8] updated figs in new dna extraction protocol --- _protocols/new-dna-extraction.md | 82 ++++++++++++++++++++++---------- 1 file changed, 58 insertions(+), 24 deletions(-) diff --git a/_protocols/new-dna-extraction.md b/_protocols/new-dna-extraction.md index 139ad96..5696d3a 100644 --- a/_protocols/new-dna-extraction.md +++ b/_protocols/new-dna-extraction.md @@ -8,45 +8,76 @@ image: /images/protocols/beer-dna-extraction.jpg 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. TrisHCl-buffer (1M, pH 7.4) +Needed consumables: +1. Yeast DNA Extraction Kit Thermo Fischer +2. TrisHCl-buffer (1M, pH 7.4) -1.0mL per sample + 50ml per sample (washing) -2. 70% EtOH (for molecular biology) +3. 70% EtOH (for molecular biology) - 1.5mL per sample -3. Isopropanol (for molecular biology) +4. Isopropanol (for molecular biology) - 600µL per sample -4. Sterile water (for molecular biology) +5. Sterile water (for molecular biology) - 50µL per sample -5. Sterile eppis (1.5mL) +6. Sterile eppis (1.5mL) - 2 per sample -6. Falcon tubes (according to beer volume) -7. Cool down centrifuge for falcon tubes -8. Switch on thermo block at 65°C +7. Falcon tubes (according to beer volume) +8. Cool down centrifuge for falcon tubes +9. Switch on thermo block at 65°C ### Before starting -Prewarm DNA Releasing Reagent A and B at 37°C for 5 min +- Prewarm DNA Releasing Reagent A and B at 37°C for 5 min +- Pre-cooled the centrifuge 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]. ### 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) - - You should carefully shake it to remove most of the CO2 -3. Transfer the beer (not the foam) into 50 ml Falcon tubes -4. Centrifuge 4000 rpm 10 min 4°C -5. Discard carefully the supernatant either by pouring the liquid phase. But anyway, be sure that the pellet remains in the Falcon. -6. Transfer 1 ml of TrisHCl-buffer (1M, pH 7.4) to the Falcon. 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 turned into a brownish color. -7. Fill up to 20ml -8. Centrifuge 4000 rpm 10 min 4°C -9. Discard supernatant -10. Resuspend the cells (with ca. 1ml 1M TrisHCl buffer pH 7.4) -11. Transfer the solution into a 1.5 ml Eppendorf tube. -12. Weigth pellets (fresh empty 1.5ml eppi as tara): 6 eppis, weights between 30mg and 60mg -13. Pool pellets from two eppis to achieve ca. 70-90mg pellet per eppi (final # of eppis: 3) +3. You should carefully shake the Erlenmeyer to remove most of 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%"} + +4. Transfer the beer (not the foam) into 50 ml Falcon 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 4000 rpm 10 min 4°C + 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): + + ![](/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 Falcon. +7. Transfer 1 ml of TrisHCl-buffer (1M, pH 7.4) to the Falcon. + + ![](/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 turned into a brownish color. + + ![](/images/protocols/beer-dna-extraction/suspend_pellet.svg){: width="85%"} + +9. Fill up to 20ml +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. + ![](/images/protocols/beer-dna-extraction/transfer_to_eppendorf.svg){: width="20%"} + +14. Weigth pellets (fresh empty 1.5ml eppi as tara): 6 eppis, weights between 30mg and 60mg +15. Pool pellets from two eppis to achieve ca. 70-90mg pellet per eppi (final # of eppis: 3) ### STEP 2: Break-down the yeast cell wall – first round +e 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. + +![](/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)* + Here: incubation times can be prolonged for difficult to lyse organisms - Suspend cells in an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. - We assumed all pellets correspond to 80 mg and added 640µl Y-PER @@ -71,10 +102,10 @@ Here: incubation times can be prolonged for difficult to lyse organisms - Transfer supernatant (only 900µl!!!!!) to a new 1.5mL centrifuge tube. -### STEP 5: Precipitate (find a non-scientific word!) the DNA +### STEP 5: Separate the DNA from other molecules (proteins) - Add 600μL isopropyl alcohol to fill tube. - Mix gently by inversion. (>20x). -- Precipitate genomic DNA by centrifuging the mixture at 13,000 × g for 10 minutes. +- Separate genomic DNA by centrifuging the mixture at 13,000 × g for 10 minutes. ### STEP 6: Wash the DNA to remove unwanted substances @@ -87,4 +118,7 @@ Here: incubation times can be prolonged for difficult to lyse organisms - Resuspend in 50μL sterile water. Pellet should solubilize completely within 5 minutes (not really). 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. +- Freeze the DNA until library preparation + +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... From a1c4e4d41ad96e95d8ded2c76c5ad4378e69677f Mon Sep 17 00:00:00 2001 From: muellert Date: Sun, 15 Mar 2020 17:02:27 +0100 Subject: [PATCH 4/8] refined the extraction protocoll --- _protocols/new-dna-extraction.md | 86 +++++++++++++++++--------------- 1 file changed, 47 insertions(+), 39 deletions(-) diff --git a/_protocols/new-dna-extraction.md b/_protocols/new-dna-extraction.md index 5696d3a..8ff6ce8 100644 --- a/_protocols/new-dna-extraction.md +++ b/_protocols/new-dna-extraction.md @@ -9,7 +9,7 @@ image: /images/protocols/beer-dna-extraction.jpg 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 +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) @@ -20,76 +20,79 @@ Needed consumables: - 50µL per sample 6. Sterile eppis (1.5mL) - 2 per sample -7. Falcon tubes (according to beer volume) -8. Cool down centrifuge for falcon tubes -9. Switch on thermo block at 65°C - +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 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]. +- 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 the size depends on the beer, its temperature and for how long it was open. +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 Falcon tubes +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 4000 rpm 10 min 4°C - 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%"} -6. Discard carefully the supernatant either by pouring the liquid phase. But anyway, be sure that the pellet remains in the Falcon. -7. Transfer 1 ml of TrisHCl-buffer (1M, pH 7.4) to 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). ![](/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 turned into a brownish color. +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 +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. +13. Transfer the solution into a 1.5 ml Eppendorf tube (eppi). ![](/images/protocols/beer-dna-extraction/transfer_to_eppendorf.svg){: width="20%"} - -14. Weigth pellets (fresh empty 1.5ml eppi as tara): 6 eppis, weights between 30mg and 60mg -15. Pool pellets from two eppis to achieve ca. 70-90mg pellet per eppi (final # of eppis: 3) +14. Centrifuge 4000 rpm 10 min 4°C +15. Discard supernatant +16. Weigh each of the 6 eppy pellets (use empty 1.5ml eppi as tara): weights of one pellet between 30mg and 60mg +17. Resolve the pellets by adding 500 μL and pipet up and down +18. Combine the solution of two eppis into one to achieve ca. 70-90mg pellet per eppi (final # of eppis: 3) +19. Centrifuge 4000 rpm 10 min 4°C +20. Discard supernatant ### STEP 2: Break-down the yeast cell wall – first round -e 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. +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)* -Here: incubation times can be prolonged for difficult to lyse organisms -- Suspend cells in an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. - - We assumed all pellets correspond to 80 mg and added 640µl Y-PER -- Mix by pipetting up and down until the mixture is homogenous +- Suspend cells: + 1. add an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. + - 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 minutes. (can be extended – SHOULD BE EXTENDED??) +- Centrifuge at 13,000 × g for 5 minutes +- Discard supernatant ### STEP 3: Break-down the yeast cell wall – second round -Here: incubation times can be prolonged for difficult to lyse organisms -- Centrifuge at 13,000 × g for 5 minutes, -- Discard supernatant, -- Add 400μL of DNA Releasing Reagent A, + +- Add 400μL of DNA Releasing Reagent A - Add 400μL of DNA Releasing Reagent B - Mix by pipetting up and down until the mixture is homogenous - Incubate at 65°C for 10 minutes. (can be extended – SHOULD BE EXTENDED??) @@ -97,28 +100,33 @@ Here: incubation times can be prolonged for difficult to lyse organisms ### STEP 4: Stop protein activity in the solution - Add 200μL of Protein Removal Reagent to mixture -- Invert several times (>20x). +- Invert eppy several times (>20x) - Centrifuge at least 13,000 × g for 5 minutes -- Transfer supernatant (only 900µl!!!!!) to a new 1.5mL centrifuge tube. +- 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 (proteins) -- Add 600μL isopropyl alcohol to fill tube. -- Mix gently by inversion. (>20x). -- Separate genomic DNA by centrifuging the mixture at 13,000 × g for 10 minutes. +### 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 × g for 10 minutes. + - The DNA be at the bottom of the eppy (pellet) +- Remove supernatant, being careful not to discard any of the pellet, which is clear and hard to see. ### STEP 6: Wash the DNA to remove unwanted substances -- Remove supernatant, being careful not to discard any of the pellet, which is clear and hard to see. -- Add 1.5mL of 70% ethanol to the pellet, invert several times (>20x). +- Add 1.5mL of 70% ethanol to the pellet +- invert eppy several times (>20x) - Centrifuge at 13,000 × g for 1 minute to wash off any residual salts or cellular debris clinging to the DNA or tube. -- Invert the tube to dry any residual ethanol before proceeding to Step 7. (took ca. 30-45min) +- 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 -- Resuspend in 50μL sterile water. Pellet should solubilize completely within 5 minutes (not really). 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. - -- 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... From c822c939d41d240df0edf040af8a2a51f989bf19 Mon Sep 17 00:00:00 2001 From: muellert Date: Mon, 16 Mar 2020 09:46:23 +0100 Subject: [PATCH 5/8] changes from Martina --- _protocols/new-dna-extraction.md | 27 ++++++++++++++------------- 1 file changed, 14 insertions(+), 13 deletions(-) diff --git a/_protocols/new-dna-extraction.md b/_protocols/new-dna-extraction.md index 8ff6ce8..af67c98 100644 --- a/_protocols/new-dna-extraction.md +++ b/_protocols/new-dna-extraction.md @@ -64,13 +64,14 @@ Needed consumables: 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 4000 rpm 10 min 4°C +14. Centrifuge 8000 rcf 5 min 4°C 15. Discard supernatant -16. Weigh each of the 6 eppy pellets (use empty 1.5ml eppi as tara): weights of one pellet between 30mg and 60mg -17. Resolve the pellets by adding 500 μL and pipet up and down -18. Combine the solution of two eppis into one to achieve ca. 70-90mg pellet per eppi (final # of eppis: 3) -19. Centrifuge 4000 rpm 10 min 4°C -20. 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 @@ -83,10 +84,10 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell - Suspend cells: 1. add an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. - - We assume all pellets correspond to 80 mg and added 640µl Y-PER + - For symplification 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 minutes. (can be extended – SHOULD BE EXTENDED??) -- Centrifuge at 13,000 × g for 5 minutes +- Incubate at 65°C for 10 - 30 minutes. +- Centrifuge at 13,000 rcf for 5 minutes - Discard supernatant @@ -95,13 +96,13 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell - Add 400μL of DNA Releasing Reagent A - Add 400μL of DNA Releasing Reagent B - Mix by pipetting up and down until the mixture is homogenous -- Incubate at 65°C for 10 minutes. (can be extended – SHOULD BE EXTENDED??) +- 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 × g for 5 minutes +- 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 @@ -109,7 +110,7 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell ### 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 × g for 10 minutes. +- Separate DNA by centrifuging the mixture at 13,000 rcf for 10 minutes. - The DNA be at the bottom of the eppy (pellet) - Remove supernatant, being careful not to discard any of the pellet, which is clear and hard to see. @@ -117,7 +118,7 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell ### 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 × g for 1 minute to wash off any residual salts or cellular debris clinging to the DNA or tube. +- 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) From c8a27ea577c0d8bc40f8a9aa83797d2b1f63dbe9 Mon Sep 17 00:00:00 2001 From: teresa-m Date: Tue, 18 Aug 2020 13:48:49 +0200 Subject: [PATCH 6/8] added last changes --- Makefile | 0 _layouts/base.html | 0 _protocols/new-dna-extraction.md | 0 css/print.scss | 0 images/protocols/beer-dna-extraction/yeast_cell.svg | 0 js/scripts.js | 0 6 files changed, 0 insertions(+), 0 deletions(-) mode change 100644 => 100755 Makefile mode change 100644 => 100755 _layouts/base.html mode change 100644 => 100755 _protocols/new-dna-extraction.md mode change 100644 => 100755 css/print.scss mode change 100644 => 100755 images/protocols/beer-dna-extraction/yeast_cell.svg mode change 100644 => 100755 js/scripts.js diff --git a/Makefile b/Makefile old mode 100644 new mode 100755 diff --git a/_layouts/base.html b/_layouts/base.html old mode 100644 new mode 100755 diff --git a/_protocols/new-dna-extraction.md b/_protocols/new-dna-extraction.md old mode 100644 new mode 100755 diff --git a/css/print.scss b/css/print.scss old mode 100644 new mode 100755 diff --git a/images/protocols/beer-dna-extraction/yeast_cell.svg b/images/protocols/beer-dna-extraction/yeast_cell.svg old mode 100644 new mode 100755 diff --git a/js/scripts.js b/js/scripts.js old mode 100644 new mode 100755 From dff8edaa59b961edd2bcf7e30ce81aa9c5b5f2af Mon Sep 17 00:00:00 2001 From: teresa-m Date: Tue, 18 Aug 2020 13:50:00 +0200 Subject: [PATCH 7/8] added gemfile.lock --- Gemfile.lock | 0 1 file changed, 0 insertions(+), 0 deletions(-) mode change 100644 => 100755 Gemfile.lock diff --git a/Gemfile.lock b/Gemfile.lock old mode 100644 new mode 100755 From 88f976de9a61454af4679f1194728b3ce4700c9d Mon Sep 17 00:00:00 2001 From: teresa-m Date: Sat, 19 Dec 2020 16:54:16 +0100 Subject: [PATCH 8/8] added Masakos bio explanations --- _protocols/new-dna-extraction.md | 24 +++++++++++++++++++----- 1 file changed, 19 insertions(+), 5 deletions(-) diff --git a/_protocols/new-dna-extraction.md b/_protocols/new-dna-extraction.md index af67c98..c249ce7 100755 --- a/_protocols/new-dna-extraction.md +++ b/_protocols/new-dna-extraction.md @@ -83,9 +83,17 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell *A yeast cell - Frankie Robertson, CC ASA, [Wikimedia](https://en.wikipedia.org/wiki/File:Yeast_cell_english.svg)* - Suspend cells: - 1. add an appropriate amount of the Y-PER Reagent. Scale the amount of Y-PER Reagent accordingly, maintaining a ratio of 8μL/1mg pellet. - - For symplification 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 + + 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 @@ -95,6 +103,9 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell - 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. @@ -111,8 +122,11 @@ Now, we want to get the DNA out the yeast. The DNA is well protected by the cell - 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 be at the bottom of the eppy (pellet) -- Remove supernatant, being careful not to discard any of the pellet, which is clear and hard to see. +- 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