Team:TU Delft/Notebook
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.
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.
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.
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]
