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Team:York UK/Notebook.html
From 2013.igem.org
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<p><img src="https://static.igem.org/mediawiki/2013/4/43/Gel-mtrA.jpg" width="340" height="150" alt="mtrAtrial" align="left" >The gel. Three different temperatures (51-61˚C)- all work. 1% agarose dyed with SYBR-Safe, however, due to poor practice the ladder is indistinguishable.</p> <p>At this point Jonas arrived at the realisation that as long as the primers are specific enough, there is no point in doing PCR trials (silly Jonas). Therefore most of the PCRs done this day forth use a universal 58˚C temperature.</p> | <p><img src="https://static.igem.org/mediawiki/2013/4/43/Gel-mtrA.jpg" width="340" height="150" alt="mtrAtrial" align="left" >The gel. Three different temperatures (51-61˚C)- all work. 1% agarose dyed with SYBR-Safe, however, due to poor practice the ladder is indistinguishable.</p> <p>At this point Jonas arrived at the realisation that as long as the primers are specific enough, there is no point in doing PCR trials (silly Jonas). Therefore most of the PCRs done this day forth use a universal 58˚C temperature.</p> | ||
<h2>July 27th</h2><p>This is the first day that we actually got some positive outcome from our cloning. Or so we thought.</p><p>All the fragments of our mtr complex were once again amplified via PCR but this time using Phusion polymerase for high fidelity (Thermo Fisher). Gel confirmed that everything went fine and specifically. Using QIAquick PCR purification kit (Qiagen) we clean up the PCR product, then measure the concentrations using NanoDrop1000, do the required calculations and then proceed to the main reaction:</p><p>In 60 minutes at 50 ˚C we joined all of the 5 fragments using the Gibson Assembly Master Mix provided by NEB. Confirmed by a PCR.</p><p> <img src="https://static.igem.org/mediawiki/2013/5/54/Gel-mtrGibson1.jpg" width="120" height="250" alt="mtrGibson1" align="left" >The PCR was not fully specific but it is unclear whether these fragments formed during Gibson reaction (main fragment joining sites are identical RBS) or because the temperature used was too low (51˚C & 58˚C). 3rd and 4th lanes are negative controls. 1% agarose dyed with SYBR-Safe, <a href="http://www.york-bio.com/QStep4.jpg">QStep4 ladder.</a></p> | <h2>July 27th</h2><p>This is the first day that we actually got some positive outcome from our cloning. Or so we thought.</p><p>All the fragments of our mtr complex were once again amplified via PCR but this time using Phusion polymerase for high fidelity (Thermo Fisher). Gel confirmed that everything went fine and specifically. Using QIAquick PCR purification kit (Qiagen) we clean up the PCR product, then measure the concentrations using NanoDrop1000, do the required calculations and then proceed to the main reaction:</p><p>In 60 minutes at 50 ˚C we joined all of the 5 fragments using the Gibson Assembly Master Mix provided by NEB. Confirmed by a PCR.</p><p> <img src="https://static.igem.org/mediawiki/2013/5/54/Gel-mtrGibson1.jpg" width="120" height="250" alt="mtrGibson1" align="left" >The PCR was not fully specific but it is unclear whether these fragments formed during Gibson reaction (main fragment joining sites are identical RBS) or because the temperature used was too low (51˚C & 58˚C). 3rd and 4th lanes are negative controls. 1% agarose dyed with SYBR-Safe, <a href="http://www.york-bio.com/QStep4.jpg">QStep4 ladder.</a></p> | ||
| - | <br clear=all><p><h2>Before August 11th</h2></p><p>During this time we tried putting all of our genes inside the pSB1C3 iGEM plasmid but with little luck. Everything seemed to be working fine up until the point when we realised that colony PCRs are not so trustworthy. It appeared clear that most of the colony PCRs came out as false positives after we decided to run the PCR on already purified plasmids. However, some samples were already on their way for sequencing (thinking that we actually got something within our pSB1C3 backbones we sent them). The sequencing results gave us an answer: we planned it all wrong! Graphical summary:</p><p><img src="https://static.igem.org/mediawiki/2013/4/43/Themistake.jpg" width="680" height="307" alt="mistake"</p><p>So all of our parts that were to be put inside the iGEM plasmid only had XbaI and SpeI cutting sites, | + | <br clear=all><p><h2>Before August 11th</h2></p><p>During this time we tried putting all of our genes inside the pSB1C3 iGEM plasmid but with little luck. Everything seemed to be working fine up until the point when we realised that colony PCRs are not so trustworthy. It appeared clear that most of the colony PCRs came out as false positives after we decided to run the PCR on already purified plasmids. However, some samples were already on their way for sequencing (thinking that we actually got something within our pSB1C3 backbones we sent them). The sequencing results gave us an answer: we planned it all wrong! Graphical summary:</p><p><img src="https://static.igem.org/mediawiki/2013/4/43/Themistake.jpg" width="680" height="307" alt="mistake"</p><p>So all of our parts that were to be put inside the iGEM plasmid only had XbaI and SpeI cutting sites, but we were unaware that they actually had complementary sticky ends and it was nearly impossible to ligate something in between.</p><p>We fixed it with new primers which add the remaining part of the prefix and suffix. However, we lost 2 weeks worth of productive labwork.</p> |
<h2>August 12th</h2><p>So after receiving the new primers we continue working. This time we're using Gibson's assembly to directly insert our mtr part into pSB1C3, this allows us to skip any problems that might occur during digestion or ligation.Note that during design of these primers we decided to produce two different mtr parts, one of which would lack the promoter and the first RBS(crR12) which we thought might not be as convenient for other teams.</p> | <h2>August 12th</h2><p>So after receiving the new primers we continue working. This time we're using Gibson's assembly to directly insert our mtr part into pSB1C3, this allows us to skip any problems that might occur during digestion or ligation.Note that during design of these primers we decided to produce two different mtr parts, one of which would lack the promoter and the first RBS(crR12) which we thought might not be as convenient for other teams.</p> | ||
<p>So today we tried amplifying mtrAB(from the previous Gibson), mtrC(from previous PCR, should have used the genome) and pSB1C3 backbone(the linearised plasmid from iGEM HQ). Unfortunately, the backbone PCR failed.</p> | <p>So today we tried amplifying mtrAB(from the previous Gibson), mtrC(from previous PCR, should have used the genome) and pSB1C3 backbone(the linearised plasmid from iGEM HQ). Unfortunately, the backbone PCR failed.</p> | ||
Revision as of 07:18, 27 September 2013
Overview
My part of the team - Gintare, Evaldas and Jonas - were responsible for cloning of the mtr complex, gold sensing device and preparing peptides for expression.
July 7th
Making of chemically competent DH5α cells. Protocol can be found in our protocols section.
July 10th
Purification of genomic DNA from Shewanella oneidensis and Salmonella sp.
July 25th
After a long break we finally started working on the actual assembly of parts - the mtr complex. Today Jonas ran a trial PCR on mtrB and mtrC part of the complex both of which are around the size of 2kb. GoTaq polymerase (Promega) was used, this is the polymerase we generally use for trials and colony PCRs as fidelity is not the main priority.
This is the gel from the PCR. Temperatures ranged 46-71˚C. It appears that the primers are sufficiently specific at even low temperatures. 1% agarose, SYBR-Safe(Invitrogen), QStep4 ladder.
July 26th
Trial PCR again, this time for three small (~500bp) synthetic DNA fragments - gBlocks (IDT), these fragments are highlighted below on the diagram as well as the overall view of our mtr gene. 
The gel. Three different temperatures (51-61˚C)- all work. 1% agarose dyed with SYBR-Safe, however, due to poor practice the ladder is indistinguishable.
At this point Jonas arrived at the realisation that as long as the primers are specific enough, there is no point in doing PCR trials (silly Jonas). Therefore most of the PCRs done this day forth use a universal 58˚C temperature.
July 27th
This is the first day that we actually got some positive outcome from our cloning. Or so we thought.
All the fragments of our mtr complex were once again amplified via PCR but this time using Phusion polymerase for high fidelity (Thermo Fisher). Gel confirmed that everything went fine and specifically. Using QIAquick PCR purification kit (Qiagen) we clean up the PCR product, then measure the concentrations using NanoDrop1000, do the required calculations and then proceed to the main reaction:
In 60 minutes at 50 ˚C we joined all of the 5 fragments using the Gibson Assembly Master Mix provided by NEB. Confirmed by a PCR.
The PCR was not fully specific but it is unclear whether these fragments formed during Gibson reaction (main fragment joining sites are identical RBS) or because the temperature used was too low (51˚C & 58˚C). 3rd and 4th lanes are negative controls. 1% agarose dyed with SYBR-Safe, QStep4 ladder.
Before August 11th
During this time we tried putting all of our genes inside the pSB1C3 iGEM plasmid but with little luck. Everything seemed to be working fine up until the point when we realised that colony PCRs are not so trustworthy. It appeared clear that most of the colony PCRs came out as false positives after we decided to run the PCR on already purified plasmids. However, some samples were already on their way for sequencing (thinking that we actually got something within our pSB1C3 backbones we sent them). The sequencing results gave us an answer: we planned it all wrong! Graphical summary:
So all of our parts that were to be put inside the iGEM plasmid only had XbaI and SpeI cutting sites, but we were unaware that they actually had complementary sticky ends and it was nearly impossible to ligate something in between.
We fixed it with new primers which add the remaining part of the prefix and suffix. However, we lost 2 weeks worth of productive labwork.
August 12th
So after receiving the new primers we continue working. This time we're using Gibson's assembly to directly insert our mtr part into pSB1C3, this allows us to skip any problems that might occur during digestion or ligation.Note that during design of these primers we decided to produce two different mtr parts, one of which would lack the promoter and the first RBS(crR12) which we thought might not be as convenient for other teams.
So today we tried amplifying mtrAB(from the previous Gibson), mtrC(from previous PCR, should have used the genome) and pSB1C3 backbone(the linearised plasmid from iGEM HQ). Unfortunately, the backbone PCR failed.
We thought that perhaps dimers of the primers formed as the sequences of prefix and suffix are not that different. 1% agarose dyed with SYBR-Safe, 1kb ladder(Invitrogen)(right).
However, after playing a bit with DMSO and annealing temperatures we were still unable to amplify it until we changed the template. We took a random iGEM part we found in the fridge and tried the PCR on it and it worked. Conclusion: iGEM HQ should provide more information about their plasmid linearisation and how were the ends modified.
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The University of York
The University of York is Lorem ipsum dolor sit amet, consectetur adipiscing elit. Morbi in sem sodales, viverra nunc id, interdum tortor. Sed urna augue, dictum eget justo ut, dictum elementum massa. Nunc eu metus nunc. Aenean tempor sit amet quam accumsan vulputate. Curabitur nec tempus quam, quis fermentum leo. In laoreet venenatis arcu, sit amet elementum leo dignissim ut. Aenean id elementum nulla. Ut velit neque, lobortis id mollis quis, luctus sed tortor. Nam aliquam vitae orci et pharetra. Nunc ut metus in orci venenatis fermentum. Suspendisse placerat est purus, sagittis vehicula elit fermentum vitae. Aenean eleifend, odio sit amet semper dictum, dolor tortor feugiat nisl, ac ornare enim nisi sit amet purus. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Pellentesque sed turpis pretium, feugiat eros sit amet, consequat ligula.
