Team:Virtus-Parva Mexico/Notebook
From 2014.igem.org
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- | + | <!-- Bitacora Biologia --> | |
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- | <h1><i>Biology Notebook</i></h1></div> | + | <h1><i>Biology Notebook</i></h1> |
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<h5><b> 29/05/2014</b></h5></p> | <h5><b> 29/05/2014</b></h5></p> | ||
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<li>Mix TE buffer</li> | <li>Mix TE buffer</li> | ||
<li>Sterilize solutions</li></ul> | <li>Sterilize solutions</li></ul> | ||
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<h5><b> 30/05/2014</b></h5></p> | <h5><b> 30/05/2014</b></h5></p> | ||
<br> After the Tris hydration, a pH of 6.55 was obtained. What was to follow was to achieve a pH of 8 by neutralizing with NaOH. | <br> After the Tris hydration, a pH of 6.55 was obtained. What was to follow was to achieve a pH of 8 by neutralizing with NaOH. | ||
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<p> Sudden realization came when the pHmeter was reviewed; it wasn’t working, so the pH was, in fact, much higher than the one figured on display. The next step was to acidify by dropwise adding of HCl.</p> | <p> Sudden realization came when the pHmeter was reviewed; it wasn’t working, so the pH was, in fact, much higher than the one figured on display. The next step was to acidify by dropwise adding of HCl.</p> | ||
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<td>8.02</td> | <td>8.02</td> | ||
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<p> In order to dissolve the EDTA in water, it was needed to raise the pH up to 8 (it was at 3.83)</p> | <p> In order to dissolve the EDTA in water, it was needed to raise the pH up to 8 (it was at 3.83)</p> | ||
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<br> Both solutions remained at room temperature after sterilizing.</p> | <br> Both solutions remained at room temperature after sterilizing.</p> | ||
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<li>Transfer into Eppendorf tubes</li></ul> | <li>Transfer into Eppendorf tubes</li></ul> | ||
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<p><h5><b> 05/09/2014</b></h5></p> | <p><h5><b> 05/09/2014</b></h5></p> | ||
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<li>Electrophoresis using DNA as λ marker with an integrated colorant to the system</li> | <li>Electrophoresis using DNA as λ marker with an integrated colorant to the system</li> | ||
<li>Sonication of the complex with a λ marker and electrophoresis evaluation</li></ul> | <li>Sonication of the complex with a λ marker and electrophoresis evaluation</li></ul> | ||
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Revision as of 14:43, 17 October 2014
Notebooks
Inorganic Notebook
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03/07/2014
04/07/2014
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07/07/2014
SiO2, n = 1.46 2.42 + 1.46 = 3.15 08/07/2014
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09/07/2014
10/07/2014
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11/07/2014The samples were once again washed with water, these samples easily precipitated in water, contrary to previous samples. Code names for these samples were NB2_(1-3) To help the precipitation, a little amount of acetone was added, this was also done for sample SB2’s aliquots. The samples were magnetically decanted and dried under vacuum for 4 hours. |
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15/07/14Samples were taken of pure magnetite, silanized magnetite (NB2) and magnetite coated with silica and silane (SB2_1 and SB2_3) for its characterization in IR for solids. Comparing the spectra given by the IR of the pure magnetite and silanized magnetite (SB2 and NB2) we were able to distinguish a peak at 990.2 cm-1 corresponding to a Si-O bond, confirming the correct silanization of the magnetite, although the amino group couldn’t be identified. Similar results were achieved for samples SB2_1 and SB2_3, however, this peak was not decisive to know if the sample was correctly functionalized giving the fact that the samples are coated with silica. |
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Biology Notebook
29/05/2014For the first day of wetlab, we prepared the solutions we would use throughout our project.
After, we prepared a solution of EDTA 0.5 M by adding 3.65 grams in 25 mL of water. The Tris 1M solution took 3.02 grams in 25 mL water. These would be further diluted later on. A mistake was made within the calculations, a redo would have to be performed. The correct amount of EDTA and Tris were weighed and left for later. The day lasted longer than anticipated, the task list involved:
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30/05/2014After the Tris hydration, a pH of 6.55 was obtained. What was to follow was to achieve a pH of 8 by neutralizing with NaOH.
Sudden realization came when the pHmeter was reviewed; it wasn’t working, so the pH was, in fact, much higher than the one figured on display. The next step was to acidify by dropwise adding of HCl.
In order to dissolve the EDTA in water, it was needed to raise the pH up to 8 (it was at 3.83)
The first of many sterilizations took place (1 box of blue tips, 1 box of yellow tips, TE Buffer and EDTA
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03/06/2014DNA extraction of E. Coli began:
The vacuum chamber was cleaned with EtOH and kept under UV light for 20 minutes. To each tube, 567 µl of TE and 30 µl of proteinase K 20 mg/ml were added. This was incubated in oven at 37 °C for an hour, then placed in the refrigerator
09/06/2014As proteinase K supply was running low, a distinct method had to be pursued.
The to-do list for the following day was as follows:
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10/06/2014Part of the team centrifuged a day before. Re-centrifugation lasted for 10 minutes. The supernatant was cleared off. The Epperndorfs were left to dry out for about half an hour. After adding 0.5 ml of TE and two volumes of absolute EtOH, the DNA strings were not visible. We centrifuged at 1300 rpm, then a volume of 50 µl TE was added to ‘A’ labelled tubes and 200 µl to ‘B’ labelled tubes. We added DNA extracted with the first protocol to the ‘A’ labelled tubes and these were re-labelled ADN-1, labels of C-1 and C-2 were conscripted.
Electrophoresis was run on DNA to check in indeed DNA was present.
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11/06/2014Ran electrophoresis over 80 V for approximately an hour and a half. 07/08/2014The protein finally arrived. WetLab time was on the go. We re-suspended the protein and prepared EDTA and Tris/acetate.
Magnetite was dispersed in anhydric toluene followed by the addition of the resuspended protein with some triethylamine and finally glutaraldehyde was added as a coupling agent 15/08/2014Alpha samples were divided into αG (G for glutaraldehyde) and αSG (without G), β samples were divided in the same way as α. Both G samples (α & β) were washed 3 times with PBS and some glutaraldehyde. The SG samples were both washed 3 times with pure PBS. UV spectra were taken for characterization. 19/08/2014Proceedings to prepare TAE for electrophoresis using TRIS, acetic acid and EDTA were performed. Agarose gel was prepared with distilled water and agarose, a loading buffer was prepared with glycerol at 10%. The electrophoresis revealing did not quite displayed desired results, a new run had to be made. |
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28/08/2014DNA extraction was performed anew:
28/08/2014Transformation and preparation of DH5α was done. CaCl2 was prepared to make competent cells. For a 200 ml solution, 54 grams of CaCl2 were added. Also, chloramphenicol was needed, 34 micrograms for each 250 microliters. 500 ml of LB broth was prepared with:
03/09/2014The protein was washed 3 times in PBS and sonicated for 10 minutes. The protein was then suspended in PBS and some magnetite was added. The samples were separated in four groups, those containing DNA and those without, and within them ones containing glutaraldehyde and the rest without. 04/09/2014Plasmid resuspension in 100 microliters TE or CaCl2 |
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