Team:Virtus-Parva Mexico/Notebook
From 2014.igem.org
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- | </tr> | + | <!-- Bitacora Biologia --> |
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+ | <div id="section" style="margin-left: 5em;"> | ||
+ | <div style="margin-top: 20px;"> | ||
+ | <h1><i>Biology Notebook</i></h1> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <p> | ||
+ | |||
+ | <table> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | |||
+ | |||
+ | <p> | ||
+ | <div align="justify"><div style="margin-left: 100px;"> | ||
+ | <h5><b> 29/05/2014</b></h5></p> | ||
+ | |||
+ | <br> For the first day of wetlab, we prepared the solutions we would use throughout our project. | ||
+ | <br> | ||
+ | <ul><li>TE Buffer Recipe:</li> | ||
+ | <ol><li>10 mM Tris, pH 8 (with HCl)</li> | ||
+ | <li>1 mM EDTA</li></ol> | ||
+ | <li>To make the CTAB/NaCl solution:</li> | ||
+ | <ol><li>4.104 g of NaCl</li> | ||
+ | <li>Slowly add 10.004 g CTAB</li> | ||
+ | <li>Add 60 ml of water</li> | ||
+ | <li>Heat at 74 °C</li></ol></ul> | ||
+ | |||
+ | |||
+ | <br> After, we prepared a solution of EDTA 0.5 M by adding 3.65 grams in 25 mL of water. | ||
+ | <br> The Tris 1M solution took 3.02 grams in 25 mL water. These would be further diluted later on. | ||
+ | <br> 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. | ||
+ | <br> The day lasted longer than anticipated, the task list involved: | ||
+ | <br> <ul type="square"><li>Hydration of the EDTA and Tris</li> | ||
+ | <li>Review Tris’ pH with potentiometer</li> | ||
+ | <li>Mix TE buffer</li> | ||
+ | <li>Sterilize solutions</li></ul> | ||
+ | |||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | </div></div> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | <tr><td> | ||
+ | <p><div align="justify"><div style="margin-left: 100px;"> | ||
+ | <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. | ||
+ | |||
+ | <p> | ||
+ | <div align="center"> | ||
+ | <table border="1" style="width:20%"> | ||
+ | <tr> | ||
+ | <th>Drops added</th> | ||
+ | <th>pH</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+10</td> | ||
+ | <td>6.64</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+20</td> | ||
+ | <td>6.70</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+20</td> | ||
+ | <td>6.76</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+20</td> | ||
+ | <td>6.97</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+15</td> | ||
+ | <td>7.0</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </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> | ||
+ | |||
+ | <p> | ||
+ | <div align="center"> | ||
+ | <table border="1" style="width:20%"> | ||
+ | <tr> | ||
+ | <th>Amount added</th> | ||
+ | <th>pH</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4 drops</td> | ||
+ | <td>11.27</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>9.54</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>8.89</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>8.54</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>8.19</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>8.02</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </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> | ||
+ | |||
+ | <p> | ||
+ | <div align="center"> | ||
+ | <table border="1" style="width:20%"> | ||
+ | <tr> | ||
+ | <th>Amount added</th> | ||
+ | <th>pH</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+10 drops</td> | ||
+ | <td>3.84</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+10 drops</td> | ||
+ | <td>3.88</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+10 drops</td> | ||
+ | <td>3.93</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+10 drops</td> | ||
+ | <td>3.95</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>4.00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>4.05</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>4.13</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>4.22</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+10 drops</td> | ||
+ | <td>4.27</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>4.45</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>4.57</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>6.00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>6.60</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+1 mL</td> | ||
+ | <td>7.39</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>+13 drops</td> | ||
+ | <td>8.01</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </p> | ||
+ | |||
+ | <p> The first of many sterilizations took place (1 box of blue tips, 1 box of yellow tips, TE Buffer and EDTA | ||
+ | <br> Both solutions remained at room temperature after sterilizing.</p> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </p> | ||
+ | </td></tr> | ||
+ | |||
+ | <tr><td> | ||
+ | <p><div align="justify"><div style="margin-left: 100px;"> | ||
+ | <h5><b> 03/06/2014</b></h5></p> | ||
+ | <br> DNA extraction of E. Coli began: | ||
+ | |||
+ | <ol><li>The bacteria was grown in an LB medium, obtaining two distinct batches.</li> | ||
+ | <li>The bacteria from the 30<sup>th</sup> was used.</li></ol> | ||
+ | |||
+ | <br> 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 | ||
+ | </p> | ||
+ | |||
+ | <p> | ||
+ | <h5><b> 09/06/2014</b></h5></p> | ||
+ | <br> As proteinase K supply was running low, a distinct method had to be pursued. | ||
+ | |||
+ | |||
+ | <ol><li>The bacteria-rich broth was centrifuged: @5000 rpm, 10 minutes, 4 ºC</li> | ||
+ | <li>5 ml of TE buffer were added, then centrifuged: @5000 rpm, 5 min, 4 °C</li> | ||
+ | <li>5 ml phenol/chloroform/isoamylic solution were added after, the formation of organic phases took place, the two lesser dense were clear, the bottom one tinted a light pink.</li> | ||
+ | <li>Vortex spun for 3 minutes.</li></ol> | ||
+ | |||
+ | |||
+ | |||
+ | <br> The to-do list for the following day was as follows: | ||
+ | <ul style="square"><li>Centrifuge at 6000 rpm, 20 min, 4 ºC</li> | ||
+ | <li>Decant the supernatant</li> | ||
+ | <li>Wash well with 0.5 ml of TE</li> | ||
+ | <li>Transfer into Eppendorf tubes</li></ul> | ||
+ | |||
+ | </p> | ||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d6/Team_Virtus_Parva_Inorganic_DLS2.png" width="120" height="auto" alt=""/> | ||
+ | </div> | ||
+ | </div> | ||
+ | </p> | ||
+ | </td></tr> | ||
+ | |||
+ | |||
+ | |||
+ | <tr><td> | ||
+ | <p><div align="justify"><div style="margin-left: 100px;"> | ||
+ | <h5><b> 10/06/2014</b></h5></p> | ||
+ | <br> Part 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. | ||
+ | <br> 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. | ||
+ | |||
+ | <ol><li>Another centrifugation: 6000 rpm, 10 min, 4 ºC</li> | ||
+ | <li>Removed supernatant</li> | ||
+ | <li>Dried out</li> | ||
+ | <li>5 ml TE added, moved to Eppendorf tubes and two volumes of absolute EtOH</li> | ||
+ | <li>Re-centrifuged at 13,000 rpm</li> | ||
+ | <li>Left to dry</li> | ||
+ | <li>Poured 400 µl TE in tube C-1</li> | ||
+ | <li>Poured C-1 into C-2</li></ol> | ||
+ | |||
+ | <br> Electrophoresis was run on DNA to check in indeed DNA was present. | ||
+ | |||
+ | <ul><li>The elaboration of the gel for electrophoresis 1x with 1% agarose:</li> | ||
+ | <ul><li>49 ml of water</li> | ||
+ | <li>1 ml TAE (50x)</li> | ||
+ | <li>0.5 gr agarose</li></ul> | ||
+ | |||
+ | <ol><li>Mixed in an Erlenmeyer flask</li> | ||
+ | <li>Heated until no crystals were seen</li> | ||
+ | <li>Poured out onto the electrophoresis plaque, left it to turn into gel.</li></ol> | ||
+ | |||
+ | <ul><li>The electrophoresis buffer, 250 ml:</li> | ||
+ | <ol><li>5 ml TAE (1x)</li> | ||
+ | <li>245 ml of water</li></ol></ul> | ||
+ | |||
+ | |||
+ | </p></td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d6/Team_Virtus_Parva_Inorganic_DLS2.png" width="120" height="auto" alt=""/> | ||
+ | </div> | ||
+ | </div> | ||
+ | </p> | ||
+ | </td></tr> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <tr><td> | ||
+ | <p><div align="justify"><div style="margin-left: 100px;"> | ||
+ | <h5><b> 11/06/2014</b></h5></p> | ||
+ | <br> Ran electrophoresis over 80 V for approximately an hour and a half. | ||
+ | |||
+ | <h5><b> 07/08/2014</b></h5></p> | ||
+ | <br> The protein finally arrived. WetLab time was on the go. We re-suspended the protein and prepared EDTA and Tris/acetate. | ||
+ | |||
+ | |||
+ | <ul><li>To prepare TAE 2M</li> | ||
+ | <ol><li>24.2 g Tris base</li> | ||
+ | <li>75 mL water</li> | ||
+ | <li>5.7 mL conc. acetic acid.</li> | ||
+ | <li>10 mL of EDTA</li></ol></ul> | ||
+ | |||
+ | <br> 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 | ||
+ | |||
+ | <h5><b> 15/08/2014</b></h5></p> | ||
+ | <br> Alpha 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. | ||
+ | |||
+ | <h5><b> 19/08/2014</b></h5></p> | ||
+ | <br> Proceedings 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. | ||
+ | |||
+ | |||
+ | </p></td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d6/Team_Virtus_Parva_Inorganic_DLS2.png" width="120" height="auto" alt=""/> | ||
+ | </div> | ||
+ | </div> | ||
+ | </p> | ||
+ | </td></tr> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <tr><td> | ||
+ | <p><div align="justify"><div style="margin-left: 100px;"> | ||
+ | <h5><b> 28/08/2014</b></h5></p> | ||
+ | <br> DNA extraction was performed anew: | ||
+ | <ol><li>E. coli was cultivated in LB broth for 24 hours at 37 °C, with constant stirring.</li> | ||
+ | <li>Pouring into Falcon tubes</li> | ||
+ | <ul><li>Tube 1. Miniprep procedure for extraction</li> | ||
+ | <li>Tube 2. E. coli in 30 ml of TE, 20 ml of isopropylic alcohol and NaOH 0.1 M were added</li></ul></ol></p> | ||
+ | |||
+ | <p><h5><b> 28/08/2014</b></h5></p> | ||
+ | <br> Transformation and preparation of DH5α was done. | ||
+ | <br> 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. | ||
+ | |||
+ | <br> 500 ml of LB broth was prepared with: | ||
+ | <ol><li>5 gr Tryptone</li> | ||
+ | <li>2.5 gr Yeast extract</li> | ||
+ | <li>0.5 gr. NaCl</li></ol></p> | ||
+ | |||
+ | <p><h5><b> 03/09/2014</b></h5></p> | ||
+ | |||
+ | <br> The 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. | ||
+ | <p><h5><b> 04/09/2014</b></h5></p> | ||
+ | <br> Plasmid resuspension in 100 microliters TE or CaCl2 | ||
+ | |||
+ | </p></td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d6/Team_Virtus_Parva_Inorganic_DLS2.png" width="120" height="auto" alt=""/> | ||
+ | </div> | ||
+ | </div> | ||
+ | </p> | ||
+ | </td></tr> | ||
+ | |||
+ | <td><tr> | ||
+ | <p><h5><b> 05/09/2014</b></h5></p> | ||
+ | |||
+ | <br> Transformation of pGLO/ PSB1C3:</li></ul> | ||
+ | <br> E. coli was cultivated in 30ml of LB broth, then stirred and transferred into falcon tubes. Passed into the centrifuge and washed with cold CaCl2, resuspended and recentrifuged. | ||
+ | |||
+ | <br> <ul><li>pGLO:</li></ul> | ||
+ | <br> E.coli was transferred into vials and resuspended pGLO was added. | ||
+ | |||
+ | <br> <ul><li>pSB1C3:</li></ul> | ||
+ | <br> Some more E.coli was transferred into vials and resuspended pSB1C3 was added. All the tubes were placed in an ice tray and then left at room temperature. These were incubated at 37Cº for 30 minutes and some samples were taken from each tube to cultivate on Petri dishes. | ||
+ | |||
+ | <p><h5><b> 09/09/2014</b></h5></p> | ||
+ | <br> <ul><li>DNA purification:</ul></li> | ||
+ | <br> In the presence of ethanol it precipitates, it then was centrifuged and the supernatant disposed of. Re-suspension on PBS followed and then passed onto an Eppendorf tube and finally adding some RNAse. | ||
+ | |||
+ | <p><h5><b> 17/09/2014</b></h5></p> | ||
+ | <br> <ul><li>Protein+DNA:</ul></li> | ||
+ | <br> Different dilutions of DNA were separated labeling samples as β and γ. These, as well, were divided with and without glutaraldehyde, and subsequently with and without DNA. We did some UV characterization for all the samples. All data was saved. | ||
+ | |||
+ | <p><h5><b> 21/09/2014</b></h5></p> | ||
+ | <br> +pGLO was cultivated in LB. | ||
+ | |||
+ | <p><h5><b> 24/09/2014</b></h5></p> | ||
+ | <br> 1ml of cultivated LB was dropped in Eppendorf tubes along with +pSBC13 | ||
+ | |||
+ | <p><h5><b> 25/09/2014</b></h5></p> | ||
+ | <br> Some washes with water of the β and γ samples, some PBS was put into the mixture as well. | ||
+ | <br> Original samples stay resuspended in water, and new samples are kept on the fridge. The samples for electrophoresis and for UV characterization were prepared. | ||
+ | |||
+ | <p><h5><b> 26/09/2014</b></h5></p> | ||
+ | <br> UV was done, the machine was set with the next parameters | ||
+ | <ul><li>Spectrum method:</li> | ||
+ | <ul><li>800-200 slow speed</li> | ||
+ | <li>Measuring mode: absorbance</li></ul></ul> | ||
+ | <br> The next methodology was followed: | ||
+ | <br> After setting the target, both cells were left running the “Baseline” script. After that, the first cell is extracted and with the help of a micropipette, the solution is mixed and the cell repositioned. Then the apparatus’ process is set to start. 2 magnetite samples were analyzed, each displaying a maximum wavelength of 310nm and 312nm, respectively. For manual normalizing, the absorbance of the maximum peak was divided, the peak previous to the fall was of 273nm with a 0.578 absorbance. This was done, too, for 1.5mg/ml and 3mg/ml samples. | ||
+ | |||
+ | <br> <ul><li>Preparation of mediums LB/CP/iPGT for a pSB1C3 promoter:</ul></li> | ||
+ | <br> First, the transformation was made. For this, E.Coli was cultured in LB broth, stirred at 37Cº, transferred into a Falcon tube for centrifuging at 1500rpm 4°C during 10 minutes, and resuspended in CaCl2. Recentrifuged again and repeat. The suspension was left on ice for half an hour. | ||
+ | <br> Another centrifuging, and another resuspension, some sample was taken and passed into an Eppendorf tube containing +pGLO. Ice resting for another 30 minutes. Then, incubation in a stove at 37Cº. | ||
+ | |||
+ | <p><h5><b> 27/09/2014</b></h5></p> | ||
+ | |||
+ | <ul><li>DNA-Purification – MiniPrep</ul></li> | ||
+ | <br> The DNA in Eppendorf tubes was centrifuged, forcing it to precipitate, then the supernatant was removed and EtOH was added in order to centrifuge again. The tube was left to dry for a few hours. The tube with the DNA was added to a previously prepared solution of PBS and RNAse. | ||
+ | <br> Placed then into the thermoblock for about 15 minutes to finish incubation.Finally, the tubes were centrifuged one last time and the sample separated in two, having two different concentrations. | ||
+ | |||
+ | <p><h5><b> 28/09/2014</b></h5></p> | ||
+ | |||
+ | <br> Some of the σ samples were prepared, one with glutaraldehyde and DNA-HU, another with glutaraldehyde and DNA; the third, with DNA-HU and the last one with DNA only. | ||
+ | |||
+ | <br> The ε samples were prepared likewise its σ counterparts. A and B samples were prepared with some nanoparticles. | ||
+ | |||
+ | <p><h5><b> 30/09/2014</b></h5></p> | ||
+ | |||
+ | <br> The A10, E10, C10, B10 samples were ran in an agarose gel at 1% and prepared to an electrophoresis run. | ||
+ | |||
+ | <p><h5><b> Tests performed</b></h5></p> | ||
+ | |||
+ | <ul><li>Electroporation by using the system on BBa_K737051 as control against psBC13 alone</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> | ||
+ | </p> | ||
+ | |||
+ | </td> | ||
+ | <td> | ||
+ | |||
+ | <p><div align="center"><div style="margin-left: 100px;"> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2014/f/fa/Team_Virtus_Parva_Inorganic_DLS.png" width="350" height="auto" alt=""/> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/d/d6/Team_Virtus_Parva_Inorganic_DLS2.png" width="120" height="auto" alt=""/> | ||
+ | </div> | ||
+ | </div> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
Revision as of 15:18, 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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