Difference between revisions of "Team:TU Delft/Notebook"

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                                             <p><strong>Lycka</strong></p>
 
                                             <p><strong>Lycka</strong></p>
 
                                             <p>Cryostocks of TOP10 cobntaining E0840 and K1149051, respectively Stored at -80 degrees.</p>
 
                                             <p>Cryostocks of TOP10 cobntaining E0840 and K1149051, respectively Stored at -80 degrees.</p>
 +
                                            <p>Digestion of backbones pSB1C3, pSB4A5 and all gBlocks with EcorI and PstI.</p>
 
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                                       </div>
 
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                                    <div class="row">
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                                        <h2 class="title-style-2">14th July</h2>
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                                        <div class="col-md-12">
 +
                                            <p><strong>Lycka</strong></p>
 +
                                            <p>Gel purification of digested backbones. Dissolved in nuclease free water, stored at -20 degrees.</p>
 +
                                            <p>Ligation of inserts into backbones.</p>
 +
                                        </div>
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                                    </div>
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                                    <!-- end activity-->
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                                    <!-- put an activity here -->                                   
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                                    <div class="row">
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                                        <h2 class="title-style-2">18th July</h2>
 +
                                        <div class="col-md-12">
 +
                                            <p><strong>Lycka</strong></p>
 +
                                            <p>Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.</p>
 +
                                        </div>
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                                    </div>
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                                    <!-- end activity-->
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                                    <!-- put an activity here -->                                   
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                                    <div class="row">
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                                        <h2 class="title-style-2">19th July</h2>
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                                        <div class="col-md-12">
 +
                                            <p><strong>Lycka</strong></p>
 +
                                            <p>Transformation of biobricks from the registry kit of 2015, since the ones from 2016 yielded no colonies. Positive control: csgA. Negative control: water. Biobricks: J23100, J23108, J23105, J23117, J23113. Overnight cultivation yielded no result.</p>
 +
                                        </div>
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                                    </div>
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                                    <!-- end activity-->
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                                    <div class="row">
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                                        <h2 class="title-style-2">20th July</h2>
 +
                                        <div class="col-md-12">
 +
                                            <p><strong>Lycka and Tessa</strong></p>
 +
                                            <p>Transformation of biobricks ligated by Maria (19th of July). After overnight culture the following plates contained colonies. pSB1C3: INP_Sil_Sdom, OmpA_Sil_Taur, Sil_Sdom, SulA, BolA_ind, BolA_con, phaP. pSB4A5: BolA_con. The ones with a negative result were ligated again the next day.</p>
 +
                                        </div>
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                                    </div>
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                                    <!-- end activity-->
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                                    <!-- put an activity here -->                                   
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                                    <div class="row">
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                                        <h2 class="title-style-2">21th July</h2>
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                                        <div class="col-md-12">
 +
                                            <p><strong>Lycka</strong></p>
 +
                                            <p>Ligation of mKate, mVenus, mCerulean, OmpA_Sil_Sdom and LacI into pSB1C3. Ligation of OmpA_Sil_Taur, OmpA_Sil_Sdom, Sil_Sdom, SulA and BolA_ind into pSB4A5. Left at room temperature overnight.</p>
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                                        </div>
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                                    <div class="row">
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                                        <h2 class="title-style-2">22th July</h2>
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                                        <div class="col-md-12">
 +
                                            <p><strong>Lycka</strong></p>
 +
                                            <p>Transformation of ligation products from yesterday. Cells containing backbones pSB1C3 or pSB4A5 were plated on plates supplemented with chloramphenicol or ampicilin, respectively. After overnight cultivation the following plates contained colonies: mKate, mVenus, mCerulean, OmpA_Sil_Sdom, Sil_Sdom, SulA, BolA_ind.</p>
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                                        </div>
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Revision as of 12:24, 29 August 2016

iGEM TU Delft

Notebook

Workspace

Our lab

We have our own iGEM TU Delft lab in the new Applied Science building on the edge of the TU Delft campus. It is classified as an ML-1 lab, the lowest safety level to work with modified organisms, which is enough for our experiments. Apart from this lab, we are also working in an optical lab, which is also ML-1 classified. In here built our own laser set-up.

Our office

Our office is our homebase for when we're not working in the lab. Here we work on things like the safety tool, the wiki, the modeling, and processing our results. This summer the Bionanoscience department moved to a new building, so it took some time before we had our own office. When we finally got our office, we quickly made it our home. Next to our office, there is a meeting room, where we have a weekly meeting with our TA’s and PI’s to keep everyone up to date and discuss problems we might encounter.

Lab safety

Our lab is classified the lowest safety level (level 1), meaning that our experiments involve low to no risk. All the members of the team have successfully completed the following safety tests: Lab safety test General safety test of the building we currently work in Biological safety test for ML-1 lab General safety test of the building we worked in before June All the members of the team have received safety training, including: Introduction to sterile working General lab training (using a PCR machine, making gels, etc.) General safety information, regarding contact persons and locations The safety of our experiments was supervised by Erwin van Rijn (Safety Manager of the lab) and Jeremie Capoulade (Safety Manager of the lasers). The supplies we needed in the lab were provided with the help of our instructor Esengül Yildirim. The research has been conducted with respect to the regulations of biosafety for The Netherlands, that can be found here.

Day Notes

DATE

participants

Notes

DATE

participants

Notes

4th July 2016

Lycka

Digestion gBlocks mVenus and mKate with EcoRI and PstI.

Made liquid culture of strain containing pSB4A5 bakcbone with RFP.

5th July 2016

Lycka

Ligation of mKate into backbone pSB1C3.

Transformation of ligation product mKate and the following biobricks: K1149051 (a kind gift from Imperial College), E0840, J23100, J23108, J23105, J23117, J23113 into E. coli TOP10. Cells were plated on agar containing LB medium supplemented with chloramphenicol.

6th July 2016

Lycka

Stocked primers VF2 and VR: storage stock (100µM) and working stock (10µM).

Colony PCR of transformants from yesterday with primers VF2 and VR followed by gel electrophoresis. Gel picture yielded no bands. This might be attributed to a fault in the transformation.

11th July 2016

Lycka

Transformation of registry biobrick J23113 as a test if the transformation works. Last year's biobrick csgA was used as a positive control. After overnight cultivation, positive control yields many colonies, J23113 yields none.

12th July 2016

Lycka

Measure DNA concentration of biobricks from registry by nanodrop.

Nanodrop

Product Concentration (ng/µl)
J23113 89.3
J23117 91.7
J23105 89.3
J23108 92.7
J23100 93.4
E0840 81.9

As can be seen from the table, there is DNA presence in the samples. Therefore, something else should be causing the transformations not to work.

13th July 2016

Lycka

Cryostocks of TOP10 cobntaining E0840 and K1149051, respectively Stored at -80 degrees.

Digestion of backbones pSB1C3, pSB4A5 and all gBlocks with EcorI and PstI.

14th July

Lycka

Gel purification of digested backbones. Dissolved in nuclease free water, stored at -20 degrees.

Ligation of inserts into backbones.

18th July

Lycka

Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.

19th July

Lycka

Transformation of biobricks from the registry kit of 2015, since the ones from 2016 yielded no colonies. Positive control: csgA. Negative control: water. Biobricks: J23100, J23108, J23105, J23117, J23113. Overnight cultivation yielded no result.

20th July

Lycka and Tessa

Transformation of biobricks ligated by Maria (19th of July). After overnight culture the following plates contained colonies. pSB1C3: INP_Sil_Sdom, OmpA_Sil_Taur, Sil_Sdom, SulA, BolA_ind, BolA_con, phaP. pSB4A5: BolA_con. The ones with a negative result were ligated again the next day.

21th July

Lycka

Ligation of mKate, mVenus, mCerulean, OmpA_Sil_Sdom and LacI into pSB1C3. Ligation of OmpA_Sil_Taur, OmpA_Sil_Sdom, Sil_Sdom, SulA and BolA_ind into pSB4A5. Left at room temperature overnight.

22th July

Lycka

Transformation of ligation products from yesterday. Cells containing backbones pSB1C3 or pSB4A5 were plated on plates supplemented with chloramphenicol or ampicilin, respectively. After overnight cultivation the following plates contained colonies: mKate, mVenus, mCerulean, OmpA_Sil_Sdom, Sil_Sdom, SulA, BolA_ind.

DATE

participants

Notes

2nd August 2016

Tessa

Amplified E0840 (GFP) out of a pSB1C3-GFP plasmid using Phusion PCR. Four reactions of 50µl.

Nanodropped PCR products.

Nanodrop

Product Concentration (ng/µl)
E0840 339.6
E0840 554.1
E0840 596.9
E0840 567.6

Ran a 1% agarose gel of the PCR product.

electrophoresis

Stored product 2 and 4 in the fridge for later use.

10th August 2016

Tessa

Restricted PCR product of 3rd August, J23100 E0840, J23113 E0840 and J23117 E0840, with EcoRI-HF and PstI.

Purified restriction product.

Nanodropped purified product.

Nanodrop

Product Concentration (ng/µl)
J23100 E0840 14.7
J23113 E0840 21.3
J23117 E0840 23.3

Ligated purified product into pSB1C3.

Transformed ligation product into TOP10 strain. Using RFP as positive control and sterile MiliQ as negative control. Plated on plates with LB agar and CM.

11th August 2016

Tessa

Colony PCR'd colonies from yesterdays transformation.

Ran a 1% agarose gel of the PCR product.

electrophoresis

Transferred colony 14 (J23113 E0840 pSB1C3) and 21 (J23117 E0840 pSB1C3) into liquid LB.

12th August 2016

Tessa

Miniprepped colony 14 and 21.

Nanodropped miniprep product.

Nanodrop

Product Concentration (ng/µl)
J23113 E0840 in pSB1C3 140.6
J23117 E0840 in pSB1C3 325.6

Cryostocked colony 14 and 21.

Send miniprepped product for sequencing. [Sequence Confirmed]

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