Difference between revisions of "Team:Warwick/Experiments"

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window.location = "https://2018.igem.org/Team:Warwick/Medals";

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$("#Content1").html("<p>All Experiments can be found within our Lab Book <a href='Notebook'>here</a></p>");

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$("#Content10").html('<h2>References</h2><p>“Degradation of Estrogens by Rhodococcus zopfii and Rhodococcus equi Isolates from Activated Sludge in Wastewater Treatment Plants”, Takeshi Yoshimoto, Fumiko Nagai, Junji Fujimoto, Koichi Watanabe, Harumi Mizukoshi, Takashi Makino, Kazumasa Kimura, Hideyuki Saino, Haruji Sawada, Hiroshi Omura, Appl. Environ. Microbiol. Sep 2004, 70 (9) 5283-5289</p>');

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$("#Content1").html("<p>The experiments we carried out were altered as our project progressed into further stages and we realised that slight alterations in the protocols facilitated what we were hoping to achieve— whether it be integration, transformation or an assembly. The protocols following were those we used at the end of the timeline our adjustments. We used Benchling to document our protocols.</p>");

+

$("#Content2").html("<h2>Making Liquid Culture & Glycerol Stock</h2><br><br><p>Introduction<br>In order to maintain a stock of pure culture of bacteria, to be placed at 80 and used when needed. We made liquid culture of our B.Subtilis strain 168. This method has also been used to make liquid cultures of our E-coli containing plasmids Top10 for Plasmid A and C, Plasmid B is K-12.</p>");

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$("#Content3").html("<p>Materials<br><br>LB: 5mL<br>› Antibiotic (Kanamycin was used for our B.Sub, and Amp. was used for Top10): 5μL (if 1000x)<br>› Plate with Colonies of Bacteria<br>› Pipette tips<br>› Glycerol<br>› Orange freezer tubes</p>");

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$("#Content4").html("<p>Procedure<br><br>For Plasmids/B.Subtilis<br><br>1. Remove antibiotic from freezer, leave to thaw<br>2. Pipette 5mL of LB into falcon tube<br>3. Add 5μL of antibiotic to the falcon tube<br>4. Take up a single colony of the bacteria on a pipette tip, drop this into the falcon tube<br>5. Overnight incubation at 37℃.<br>6. take liquid culture of plasmid B out of 37C incubatomake glycerol stock of liquid culture of plasmid B by adding<br>700ul liquid culture to 300ul 50% sterile filtered glycerol in a small orange tube then put in -80C<br>7. ALTERNATIVELY: spin down in centrifuge, store pellet in freezer");

+

$("#Content5").html("<h2>PCR</h2><br><br><p>Introduction<br>PCR is used to amplify DNA etc. put this extra info in later.<br>There are three main PCR types we've been taught so far:<br> 1. Standard PCR<br>2. Colony PCR (kind of a 'hack' - remember whenever this is done, the first step is always 98C for 10mins)<br>3. Gradient PCR (where 'blocks' in the pcr machine are set to different annealing temperatures - usually done to find the best annealing temperatures for a sample before doing a proper PCR)<br>You can get 2 different volumes of PCR: 25ul and 50ul.<br>- 25ul reaction: used for screening, testing for best annealing temperature, diagnostic - basically anytime that you won't be using the DNA afterwards.<br>- 50ul reaction: used to amplify DNA up before assembly reactions.</p>");

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$("#Content6").html("<p>Materials<br><br>› PCR tube<br>› sterile distilled water - as much as is needed to bring the reaction mixture to 50ul (or 25ul, if that reaction)<br>› 2.5ul 10uM forward primer (1.25ul if 25ul reaction)<br>› 2.5ul 10uM reverse primer (1.25ul if 25ul reaction)<br>› 1ul template DNA<br>› 25ul Q5 2 x mastermix (12.5ul if 25ul reaction)</p>");

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$("#Content7").html("<p>Procedure<br><br>Standard PCR<br>1. Add all reagents in order given above (note: ALWAYS ADD Q5 LAST)<br>2. Use the neb calculator to find out annealing temperature for primers when setting up the PCR machine <br>http://tmcalculator.neb.com/#!/main</p>");

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$("#Content8").html("<h2>Multiple Colony PCR</h2><br><br><p>Introduction<br><br>You do this to check if the correct plasmid has been transformed into your cells. Once you finish the PCR you run a gel for all 11 tubes to see if what you're looking for is in there. The below was written for GvpA going into Plasmid A. Usually do a 25 ul bc you're not going to be using the PCR or gel extraction you just wanna check if your DNA is present. IMPORTANT: The below used 10 different colonies but this is only necessary because of how many colonies were there so we were skeptical about if there was contamination. You can usually just screen 5 at a time.</p>");

+

$("#Content9").html("<p>Materials<br><br>›PCR tubes (like 11)<br>› Sterile distilled water - as much as is needed to bring the reaction mixture to 50ul (or 25ul, if that reaction)<br>› 1.25ul 10uM forward primer<br>› 1.25ul 10uM reverse primer<br>› 1ul template DNA<br>› 12.5ul Q5 2 x mastermix<br>› AMP LB plate, or whatever antibiotic resistance backbone your plasmid has.");

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$("#Content10").html("<p>Procedure<br><br>Colony PCR with multiple colonies:<br>1. Make a plate with areas labelled 1-10 to grow the colonies you are PCR-ing alongside.<br> This is so that you can grow and keep the colonies which do turn out to have the correct plasmid in them.<br>2. Add 10ul of water + an individual colony to an eppendorf tube by picking it up with a pipette tip and rubbing it around the bottom of the tube.<br>3. Rub the leftover colony that's on the pipette tip onto a plate, in the area labelled 1.<br>4. Do this for 10 colonies, then place the plate in an incubator at 37.<br>NB: Make sure the plated colonies are only incubated overnight, then<br> 5. Make a water dilution of a negative control as well (step 2), no need to plate it.<br>The negative control is JDE131 without GvpA in this case because it does not have sites for the primers to anneal to.<br>6. Once the 11 colony water dilutions have been made, make a mastermix of your PCR reagents (EXCEPT Q5).Meaning a tube holding all the necessary PCR regeants for 12 PCRs (so that u have a lil extra just incase). This will involve your forward and reverse primers, make sure they are diluted to 10uM.<br>7. Vortex your 11 diluted colony tubes individually and add 1ul of your diluted colony to 11 separate PCR tubes.<br>8. Add the Q5 required for the mastermix of 12 reactions to the mastermix of your PCR reagents. You add Q5 late bc its super fragile rip<br>9. Add 24ul of the mastermix of your PCR reagents with Q5 added to your PCR tubes which contain the lil diluted colonies.<br>10. PCR under the Colony PCR settings.");

+

$("#Content11").html("<h2>Integrating them into <i>B. subtilis</i></h2><br><br><p>Introduction<br>This method is specifically for integrating into B. subtilis using cut sites inserted into plasmids which allow the insertion of the genes into the bacillus genome. In parts of our lab diary, we use transforming and integrating interchangeably, however we are fully integrating the genes into the genome, not transforming them into the cells as extra-chromosomal material or plasmids.</p>");

+

$("#Content12").html("<p>Materials<br><br>B. subtilis (PY79)<br>› 2mg of DNA you wish to integrate<br>› Transformation media<br>› LBA only plate<br>› LBA plates with appropriate antibiotic for B. subtilis colonies (Chlor and Spec in this case)<br>› 50mL falcon tube<br>› Transformation media<br>› Snap cap tubes<br>› Cuvette<br>› Glass beads");

+

$("#Content13").html("<p>Procedure<br><br>Integrating into B. subtilis<br>1. Streak B. subtilis (PY79) on a new LBA plate.<br>2. Leave the colony to grow overnight in a 37ºC to have it fresh on plate the day after.<br>3. In a 50mL falcon tube, put in transformation media. Each transformation will require of transformation media, so add (the number of transformations you expect) + (the number of control) + (how many times you expect to measure OD). For example if you want to transform 2 plasmids and you'd measure OD 2 times, you'd put 2+1+2=5mL<br>4. With an inoculation loop, take a lot of B.subtilis from the fresh plate (~10 colonies) and suspend them in the transformation media in the falcon tube, making sure there are no clumps. Try to avoid vortexing unless absolutely necessary as swirling the loop vigorously should be enough.<br>5. Grow in the shaking incubator for ~3/4 hours. If you want, measure the OD to check it has grown to a value of ~0.5. To measure the OD, 0.8mL is enough to put in the cuvette, and make sure to blank with transformation media. Make sure to take the sample from an homogeneous volume. If you want to use less volume than 0.8mL, you can use the trick of diluting it and therefore guess the OD of the original, as you've already done.<br>6. When bacillus has grown in the tube to an OD ~0.5 (or is sufficiently more translucent in the tube after 3/4h), aliquot 1mL in snap cap tubes, as many as the transformations will be as well as extra for the control experiments you are doing alongside.<br>7. In each snap cap tube add the plasmid to be transformed. Aim to have at least 2mg of DNA => e.g. if you have plasmid at a concentration of 250ng/uL you will add 8uL of plasmid. Leave one snap cap tube with 1mL bacillus and no DNA (as a negative control)<br>8. Grow in shaking incubator (at 37ºC) for 45m<br>9. In the meantime, you would have prepared plates with the correct concentration of antibiotic (spectinomycin for JDE131 plasmid, chloramphenicol for ECE174 plasmid)<br>10. When ready, spin down the snap cap tubes: 4000RPM for 3 minutes<br>11. You should have a pellet at the bottom of the tube. Carefully remove 0.9mL of supernatant, leaving the pellet with 0.1mL of supernatant. Then resuspend the pellet in the 0.1mL<br>12. Plate on appropriate plate (by using beads: first you let a few glass beads (~5) to enter the plate, then you add the 0.1mL bacillus and move the (closed) plate on the bench to make the glass beads spread the liquid homogeneously on the plate)");

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$("#Content14").html("<h2>Floating Experiments</h2><br><br><p>Introduction<br><br>Using Shapiro's protocol paper (Lakshmanan et al 2017) we looked at how helpful it might be in setting up the float experiment. Pages 2060-2061 detail how to grow E. coli mega cultures, under the section C, titled 'Production and Purification of Mega GVs'. Essentially, it seems the relevant steps are (quoting directly from the text):<br>a) 'Resuspend an aliquot of glycerol stock in 3 ml of LB medium containing 1× ampicillin, 1× chloramphenicoland 1% (wt/vol) glucose. Grow the E. coli culture to saturation (OD600 > 4).'<br>b) 'Prepare 100 ml of LB medium containing 1× ampicillin, 1× chloramphenicol and 0.2% (wt/vol) glucose, and inoculate 1 ml of the saturated E. coli culture into the broth. Grow at 37 °C for ~2 h until the OD600 value reaches 0.4 to 0.6.'<br>c) 'Induce the culture by adding 20 μM IPTG (final concentration), and grow it at 30 °C for an additional 16–24 h.'<br>We followed this protocol, with a few changes that were implemented mostly by Clare Hayes, an instrumental and key part of our team. For instance, if you wanted to try different IPTG concentrations, you would want to grow up a bit of extra culture, initially. In addition, the reason the protocol says to grow the cells in chloramphenicol is because unlike us, who used the C43 strain, they transformed the plasmid into a Rosetta 2 (DE3) strain. This strain has an extra, chloramphenicol resistant plasmid, that aids in the expression of rare codons for eukaryotic gene expression. Our cells do not have this plasmid, so we don't need to use chloramphenicol - in fact, using it would kill off our cells.");

+

$("#Content15").html("<p>Materials<br><br>› LM Media with Ampicillin (with 1 uL to 1 mL of LB culture) and Glucose (1% wt/vol glucose)<br>› 50 mL falcon tube<br>› Floating plasmid inside cells in glycerol stock (MEGA plasmid in C43 in this case)<br>› IPTG (in liquid form, but varying concentrations relative to volume)<br>› 400ml conical flask<br>› 4x 150ml conical flasks<br>› Cuvettes");

+

$("#Content16").html("<p>Procedure<br><br>1. Prepare 5 mL of media, containing a normal concentration of ampicillin (1 uL to 1 mL of LB culture), as well as 1% wt/vol glucose. Put it in a 50 mL falcon tube.<br>2. Scrape a bit of MEGA plasmid glycerol stock out of its tube, using a pipette tip. Drop this tip into the culture tube. Grow the culture up overnight in the 37C shaking incubator.<br>3. Measure the OD the next morning. Hopefully, it will have hit saturation - around an OD of 4. If it's below 4, put the tube back in the incubator to grow until saturation.<br>4. Meanwhile, make 400 mL of media, same ingredients as above (ampicillin, glucose) except at a lower glucose concentration of 0.2% (wt/vol). Put the media into a large flask. When the 5 mL culture reaches an OD of 4, pipette 4 mL of it into the flask, and shake at 37C for ~2 h.<br>5. Pull the flask right at 2 hr, or even before, and check the OD. You are aiming for an OD between 0.4 to 0.6. Keep the culture in the incubator until it reaches this range (but don't let it overgrow and go higher than 0.6 - if it gets too high, you'll have to start the whole experiment over again).<br>6. When the OD reaches 0.4 - 0.6, you'll want to induce with IPTG. First, you’ll need to divide the culture into 4 different flasks, 100 mL each. This is because, just like we did with the CFP and YFP plasmids in the plate, we want to add a different amount of IPTG to each. One of the flasks will have 0, and be our control flask, while another will have: 200 uM IPTG, 20 μM IPTG, and 2 uM IPTG (final concentrations). Just like before, you'll have to calculate to figure out how much IPTG to add to 100 mL of media to get 200 uM final concentration, and then do 1:10 serial dilutions of the stock down from there.<br>7. After adding the right amount of IPTG to each flask, change the shaking incubator temperature to 30C, and grow the flasks overnight for an additional 16–24 hours.<br>8. Look to see if the cells float! You'll also want to grow up the C43 cells as a control, so do exactly the same thing with them, except you only need to make 100 mLs of culture, not 400. Also, don't add ampicillin to them, since they aren't resistant and will die. Note: In biology, wt/vol refers to g/mL, for some reason. So, for 1% glucose solution, you need 1 gram glucose / 100 mL volume.");

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Difference between revisions of "Team:Warwick/Experiments"

Latest revision as of 03:55, 18 October 2018

Experiments

Contact Us

igem@warwick.ac.uk

Sponsors

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 		<script src="https://ajax.googleapis.com/ajax/libs/jquery/3.3.1/jquery.min.js"></script>
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 <script>
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          window.location = "https://2018.igem.org/Team:Warwick/Medals";
 }
+function showContents(){
+  $(".Contents").css("display", "block");
+}
+function hideContents(){
+  $(".Contents").css("display", "none");
+}
 function BioClick(){
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          $("#inorgIMG").css("background-color", "white");
+        $("#Content1").css("display", "block");
+        $("#Content1").html("<p>All Experiments can be found within our Lab Book <a href='Notebook'>here</a></p>");
 }
 function OrgClick(){
+        showContents();
          $("#bioIMG").css("margin", "2% 5%");
          $("#bioIMG").css("width", "10%");
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          $("#Content10").html('<h2>References</h2><p>“Degradation of Estrogens by Rhodococcus zopfii and Rhodococcus equi Isolates from Activated Sludge in Wastewater Treatment Plants”, Takeshi Yoshimoto, Fumiko Nagai, Junji Fujimoto, Koichi Watanabe, Harumi Mizukoshi, Takashi Makino, Kazumasa Kimura, Hideyuki Saino, Haruji Sawada, Hiroshi Omura, Appl. Environ. Microbiol. Sep 2004, 70 (9) 5283-5289</p>');
+        $("#Content11").css("display","none");
+        $("#Content12").css("display","none");
+        $("#Content13").css("display","none");
+        $("#Content14").css("display","none");
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+        $("#Content16").css("display","none");
 }
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          $("#inorgIMG").css("width", "12%");
          $("#inorgIMG").css("background-color", "grey");
+        $("#Content1").html("<p>The experiments we carried out were altered as our project progressed into further stages and we realised that slight alterations in the protocols facilitated what we were hoping to achieve— whether it be integration, transformation or an assembly. The protocols following were those we used at the end of the timeline our adjustments. We used Benchling to document our protocols.</p>");
+        $("#Content2").html("<h2>Making Liquid Culture & Glycerol Stock</h2><br><br><p>Introduction<br>In order to maintain a stock of pure culture of bacteria, to be placed at 80 and used when needed. We made liquid culture of our B.Subtilis strain 168. This method has also been used to make liquid cultures of our E-coli containing plasmids Top10 for Plasmid A and C, Plasmid B is K-12.</p>");
+        $("#Content3").html("<p>Materials<br><br>LB: 5mL<br>› Antibiotic (Kanamycin was used for our B.Sub, and Amp. was used for Top10): 5μL (if 1000x)<br>› Plate with Colonies of Bacteria<br>› Pipette tips<br>› Glycerol<br>› Orange freezer tubes</p>");
+        $("#Content4").html("<p>Procedure<br><br>For Plasmids/B.Subtilis<br><br>1. Remove antibiotic from freezer, leave to thaw<br>2. Pipette 5mL of LB into falcon tube<br>3. Add 5μL of antibiotic to the falcon tube<br>4. Take up a single colony of the bacteria on a pipette tip, drop this into the falcon tube<br>5. Overnight incubation at 37℃.<br>6. take liquid culture of plasmid B out of 37C incubatomake glycerol stock of liquid culture of plasmid B by adding<br>700ul liquid culture to 300ul 50% sterile filtered glycerol in a small orange tube then put in -80C<br>7. ALTERNATIVELY: spin down in centrifuge, store pellet in freezer");
+        $("#Content5").html("<h2>PCR</h2><br><br><p>Introduction<br>PCR is used to amplify DNA etc. put this extra info in later.<br>There are three main PCR types we've been taught so far:<br> 1. Standard PCR<br>2. Colony PCR (kind of a 'hack' - remember whenever this is done, the first step is always 98C for 10mins)<br>3. Gradient PCR (where 'blocks' in the pcr machine are set to different annealing temperatures - usually done to find the best annealing temperatures for a sample before doing a proper PCR)<br>You can get 2 different volumes of PCR: 25ul and 50ul.<br>- 25ul reaction: used for screening, testing for best annealing temperature, diagnostic - basically anytime that you won't be using the DNA afterwards.<br>- 50ul reaction: used to amplify DNA up before assembly reactions.</p>");
+        $("#Content6").html("<p>Materials<br><br>› PCR tube<br>› sterile distilled water - as much as is needed to bring the reaction mixture to 50ul (or 25ul, if that reaction)<br>› 2.5ul 10uM forward primer (1.25ul if 25ul reaction)<br>› 2.5ul 10uM reverse primer (1.25ul if 25ul reaction)<br>› 1ul template DNA<br>› 25ul Q5 2 x mastermix (12.5ul if 25ul reaction)</p>");
+        $("#Content7").html("<p>Procedure<br><br>Standard PCR<br>1. Add all reagents in order given above (note: ALWAYS ADD Q5 LAST)<br>2. Use the neb calculator to find out annealing temperature for primers when setting up the PCR machine <br>http://tmcalculator.neb.com/#!/main</p>");
+         $("#Content8").html("<h2>Multiple Colony PCR</h2><br><br><p>Introduction<br><br>You do this to check if the correct plasmid has been transformed into your cells. Once you finish the PCR you run a gel for all 11 tubes to see if what you're looking for is in there. The below was written for GvpA going into Plasmid A. Usually do a 25 ul bc you're not going to be using the PCR or gel extraction you just wanna check if your DNA is present. IMPORTANT: The below used 10 different colonies but this is only necessary because of how many colonies were there so we were skeptical about if there was contamination. You can usually just screen 5 at a time.</p>");
+          $("#Content9").html("<p>Materials<br><br>›PCR tubes (like 11)<br>› Sterile distilled water - as much as is needed to bring the reaction mixture to 50ul (or 25ul, if that reaction)<br>› 1.25ul 10uM forward primer<br>› 1.25ul 10uM reverse primer<br>› 1ul template DNA<br>› 12.5ul Q5 2 x mastermix<br>› AMP LB plate, or whatever antibiotic resistance backbone your plasmid has.");
+          $("#Content10").html("<p>Procedure<br><br>Colony PCR with multiple colonies:<br>1. Make a plate with areas labelled 1-10 to grow the colonies you are PCR-ing alongside.<br> This is so that you can grow and keep the colonies which do turn out to have the correct plasmid in them.<br>2. Add 10ul of water + an individual colony to an eppendorf tube by picking it up with a pipette tip and rubbing it around the bottom of the tube.<br>3. Rub the leftover colony that's on the pipette tip onto a plate, in the area labelled 1.<br>4. Do this for 10 colonies, then place the plate in an incubator at 37.<br>NB: Make sure the plated colonies are only incubated overnight, then<br> 5. Make a water dilution of a negative control as well (step 2), no need to plate it.<br>The negative control is JDE131 without GvpA in this case because it does not have sites for the primers to anneal to.<br>6. Once the 11 colony water dilutions have been made, make a mastermix of your PCR reagents (EXCEPT Q5).Meaning a tube holding all the necessary PCR regeants for 12 PCRs (so that u have a lil extra just incase). This will involve your forward and reverse primers, make sure they are diluted to 10uM.<br>7. Vortex your 11 diluted colony tubes individually and add 1ul of your diluted colony to 11 separate PCR tubes.<br>8. Add the Q5 required for the mastermix of 12 reactions to the mastermix of your PCR reagents. You add Q5 late bc its super fragile rip<br>9. Add 24ul of the mastermix of your PCR reagents with Q5 added to your PCR tubes which contain the lil diluted colonies.<br>10. PCR under the Colony PCR settings.");
+         $("#Content11").html("<h2>Integrating them into <i>B. subtilis</i></h2><br><br><p>Introduction<br>This method is specifically for integrating into B. subtilis using cut sites inserted into plasmids which allow the insertion of the genes into the bacillus genome. In parts of our lab diary, we use transforming and integrating interchangeably, however we are fully integrating the genes into the genome, not transforming them into the cells as extra-chromosomal material or plasmids.</p>");
+         $("#Content12").html("<p>Materials<br><br>B. subtilis (PY79)<br>› 2mg of DNA you wish to integrate<br>› Transformation media<br>› LBA only plate<br>› LBA plates with appropriate antibiotic for B. subtilis colonies (Chlor and Spec in this case)<br>› 50mL falcon tube<br>› Transformation media<br>› Snap cap tubes<br>› Cuvette<br>› Glass beads");
+         $("#Content13").html("<p>Procedure<br><br>Integrating into B. subtilis<br>1. Streak B. subtilis (PY79) on a new LBA plate.<br>2. Leave the colony to grow overnight in a 37ºC to have it fresh on plate the day after.<br>3. In a 50mL falcon tube, put in transformation media. Each transformation will require of transformation media, so add (the number of transformations you expect) + (the number of control) + (how many times you expect to measure OD). For example if you want to transform 2 plasmids and you'd measure OD 2 times, you'd put 2+1+2=5mL<br>4. With an inoculation loop, take a lot of B.subtilis from the fresh plate (~10 colonies) and suspend them in the transformation media in the falcon tube, making sure there are no clumps. Try to avoid vortexing unless absolutely necessary as swirling the loop vigorously should be enough.<br>5. Grow in the shaking incubator for ~3/4 hours. If you want, measure the OD to check it has grown to a value of ~0.5. To measure the OD, 0.8mL is enough to put in the cuvette, and make sure to blank with transformation media. Make sure to take the sample from an homogeneous volume. If you want to use less volume than 0.8mL, you can use the trick of diluting it and therefore guess the OD of the original, as you've already done.<br>6. When bacillus has grown in the tube to an OD ~0.5 (or is sufficiently more translucent in the tube after 3/4h), aliquot 1mL in snap cap tubes, as many as the transformations will be as well as extra for the control experiments you are doing alongside.<br>7. In each snap cap tube add the plasmid to be transformed. Aim to have at least 2mg of DNA => e.g. if you have plasmid at a concentration of 250ng/uL you will add 8uL of plasmid. Leave one snap cap tube with 1mL bacillus and no DNA (as a negative control)<br>8. Grow in shaking incubator (at 37ºC) for 45m<br>9. In the meantime, you would have prepared plates with the correct concentration of antibiotic (spectinomycin for JDE131 plasmid, chloramphenicol for ECE174 plasmid)<br>10. When ready, spin down the snap cap tubes: 4000RPM for 3 minutes<br>11. You should have a pellet at the bottom of the tube. Carefully remove 0.9mL of supernatant, leaving the pellet with 0.1mL of supernatant. Then resuspend the pellet in the 0.1mL<br>12. Plate on appropriate plate (by using beads: first you let a few glass beads (~5) to enter the plate, then you add the 0.1mL bacillus and move the (closed) plate on the bench to make the glass beads spread the liquid homogeneously on the plate)");
+         $("#Content14").html("<h2>Floating Experiments</h2><br><br><p>Introduction<br><br>Using Shapiro's protocol paper (Lakshmanan et al 2017) we looked at how helpful it might be in setting up the float experiment. Pages 2060-2061 detail how to grow E. coli mega cultures, under the section C, titled 'Production and Purification of Mega GVs'. Essentially, it seems the relevant steps are (quoting directly from the text):<br>a) 'Resuspend an aliquot of glycerol stock in 3 ml of LB medium containing 1× ampicillin, 1× chloramphenicoland 1% (wt/vol) glucose. Grow the E. coli culture to saturation (OD600 > 4).'<br>b) 'Prepare 100 ml of LB medium containing 1× ampicillin, 1× chloramphenicol and 0.2% (wt/vol) glucose, and inoculate 1 ml of the saturated E. coli culture into the broth. Grow at 37 °C for ~2 h until the OD600 value reaches 0.4 to 0.6.'<br>c) 'Induce the culture by adding 20 μM IPTG (final concentration), and grow it at 30 °C for an additional 16–24 h.'<br>We followed this protocol, with a few changes that were implemented mostly by Clare Hayes, an instrumental and key part of our team. For instance, if you wanted to try different IPTG concentrations, you would want to grow up a bit of extra culture, initially. In addition, the reason the protocol says to grow the cells in chloramphenicol is because unlike us, who used the C43 strain, they transformed the plasmid into a Rosetta 2 (DE3) strain. This strain has an extra, chloramphenicol resistant plasmid, that aids in the expression of rare codons for eukaryotic gene expression. Our cells do not have this plasmid, so we don't need to use chloramphenicol - in fact, using it would kill off our cells.");
+         $("#Content15").html("<p>Materials<br><br>› LM Media with Ampicillin (with 1 uL to 1 mL of LB culture) and Glucose (1% wt/vol glucose)<br>› 50 mL falcon tube<br>› Floating plasmid inside cells in glycerol stock (MEGA plasmid in C43 in this case)<br>› IPTG (in liquid form, but varying concentrations relative to volume)<br>› 400ml conical flask<br>› 4x 150ml conical flasks<br>› Cuvettes");
+        $("#Content16").html("<p>Procedure<br><br>1. Prepare 5 mL of media, containing a normal concentration of ampicillin (1 uL to 1 mL of LB culture), as well as 1% wt/vol glucose. Put it in a 50 mL falcon tube.<br>2. Scrape a bit of MEGA plasmid glycerol stock out of its tube, using a pipette tip. Drop this tip into the culture tube. Grow the culture up overnight in the 37C shaking incubator.<br>3. Measure the OD the next morning. Hopefully, it will have hit saturation - around an OD of 4. If it's below 4, put the tube back in the incubator to grow until saturation.<br>4. Meanwhile, make 400 mL of media, same ingredients as above (ampicillin, glucose) except at a lower glucose concentration of 0.2% (wt/vol). Put the media into a large flask. When the 5 mL culture reaches an OD of 4, pipette 4 mL of it into the flask, and shake at 37C for ~2 h.<br>5. Pull the flask right at 2 hr, or even before, and check the OD. You are aiming for an OD between 0.4 to 0.6. Keep the culture in the incubator until it reaches this range (but don't let it overgrow and go higher than 0.6 - if it gets too high, you'll have to start the whole experiment over again).<br>6. When the OD reaches 0.4 - 0.6, you'll want to induce with IPTG. First, you’ll need to divide the culture into 4 different flasks, 100 mL each. This is because, just like we did with the CFP and YFP plasmids in the plate, we want to add a different amount of IPTG to each. One of the flasks will have 0, and be our control flask, while another will have: 200 uM IPTG, 20 μM IPTG, and 2 uM IPTG (final concentrations). Just like before, you'll have to calculate to figure out how much IPTG to add to 100 mL of media to get 200 uM final concentration, and then do 1:10 serial dilutions of the stock down from there.<br>7. After adding the right amount of IPTG to each flask, change the shaking incubator temperature to 30C, and grow the flasks overnight for an additional 16–24 hours.<br>8. Look to see if the cells float! You'll also want to grow up the C43 cells as a control, so do exactly the same thing with them, except you only need to make 100 mLs of culture, not 400. Also, don't add ampicillin to them, since they aren't resistant and will die. Note: In biology, wt/vol refers to g/mL, for some reason. So, for 1% glucose solution, you need 1 gram glucose / 100 mL volume.");
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