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

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$("#Content5").html("<h2>PCR</h2> Introduction PCR is used to amplify DNA etc. put this extra info in later. There are three main PCR types we've been taught so far: 1. Standard PCR 2. Colony PCR (kind of a 'hack' - remember whenever this is done, the first step is always 98C for 10mins) 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) You can get 2 different volumes of PCR: 25ul and 50ul.

+

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

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- 25ul reaction: used for screening, testing for best annealing temperature, diagnostic - basically anytime that you won't be using the DNA afterwards. - 50ul reaction: used to amplify DNA up before assembly reactions.");

+

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

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$("#Content13").html("Procedure Integrating into B. subtilis 1. Streak B. subtilis (PY79) on a new LBA plate. 2. Leave the colony to grow overnight in a 37ºC to have it fresh on plate the day after. 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 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. 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. 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. 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) 8. Grow in shaking incubator (at 37ºC) for 45m 9. In the meantime, you would have prepared plates with the correct concentration of antibiotic (spectinomycin for JDE131 plasmid, chloramphenicol for ECE174 plasmid) 10. When ready, spin down the snap cap tubes: 4000RPM for 3 minutes 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 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> Introduction 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): 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).' 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.' c) 'Induce the culture by adding 20 μM IPTG (final concentration), and grow it at 30 °C for an additional 16–24 h.' 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

+

$("#Content14").html("<h2>Floating Experiments</h2> Introduction 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): 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).' 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.' c) 'Induce the culture by adding 20 μM IPTG (final concentration), and grow it at 30 °C for an additional 16–24 h.' 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.");

−

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

@@ Line 229: / Line 229: @@
          $("#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.
+         $("#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>");
-- 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>");
@@ Line 248: / Line 247: @@
           $("#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
+          $("#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.");
-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");

Difference between revisions of "Team:Warwick/Notebook"

Revision as of 01:29, 18 October 2018

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