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| | <font size="4"><b>Activities relating to the redoxilator</b></font><br><br> | | <font size="4"><b>Activities relating to the redoxilator</b></font><br><br> |
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| - | <font size="3"><b>July 20th</b></font><br>
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| - | <p align="justify">Construction of biobricks: The restriction products were evaluated on a 1% agarose gel (see figure 2).</p><br>
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| - | [[image:DTUgel1.jpg|300px|thumb|center|]]
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| - | <p align="center"><i>Figure 2: Restriction analysis of the three biobricks.</i><br><br></p>
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| - | <font size="3"><b>July 19th</b></font><br>
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| - | <p align="justify">Construction of biobricks: The three plasmids were purified according to the protocol. Following the purification of the plasmids, a restriction analysis was performed using SpeI enzyme (10U) and incubated at 37ºC overnight.</p><br>
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| - | <font size="3"><b>July 18th</b></font><br>
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| - | <p align="justify">Construction of biobricks: Some colonies were picked from the transformation plates and made overnight cultures in liquid media.</p><br>
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| - | <font size="3"><b>July 17th</b></font><br>
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| - | <p align="justify">Construction of biobricks: Following purification, all of the PCR product was digested with <i>Xba</i>I (60U) and <i>Spe</i>I (30U). The mixtures were incubated at 37ºC. After 8 hours of incubation, more enzyme was added (<i>Xba</i>I (20 U) and <i>Spe</i>I (10U)) to ensure maximum digestion. Following enzymatic purification a gel was run to estimate the concentration of the vector and the PCR product (see figure 1).</p><br>
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| - | [[image:DTUgel2.jpg|300px|thumb|center|]]
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| - | <p align="center"><i>Figure 1: Electrophoresis gel picture of the PCR products of the three biobricks and the vector. The band 11P at 1500 bp corresponds to the vector; the three other bands at 537 bp, 207 bp and 125 bp correspond to the fragments Cln, GFP and Cln_GFP respectively.</i><br><br>
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| - | <p align="justify">To meet the 3:1 ratio of fragment to vector 12μl fragment was mixed with 2μl vector, 2μl ligase buffer (10x), 1μl ligase, 3 μl Mq H20. The ligation of the fragment and the vector was carried out on PCR machine using a 3:1 ratio for 1.5 hours using the idea by Lund, A. H., M. Duch, and F. S. Pedersen. 1996. Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res. 24:800-801. doi:l50400 [pii].
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| - | The final product was finally transformed into competent E coli cells.</p><br>
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| - | <font size="3"><b>July 16th</b></font><br>
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| - | <p align="justify">Construction of biobricks: The 357bp Cln2 fragment was amplified by PCR using the primers BGHA351/BGHA362 and Phusion polymerase. Primers BGHA368/369 were used to amplify the GFP fragment and finally, primers BGHA368/353 were used to amplify the fragment containing the GFP fused with the Cln. Following amplification the PCR products were run on a 1% agarose gel and purified The primers added tails to the PCR product containing an <i>Xba</i>I site in the forward primer and a <i>Spe</i>I in the reverse primer to meet the “Silver fusion” standard BBF RFC 23.<br>
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| - | <a href="http://dspace.mit.edu/bitstream/handle/1721.1/32535/PhillipsSilverFusion.pdf?sequence=1">Article: "A New Biobrick Assembly Strategy Designed for Facile Protein Engineering"</a><br>
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| - | <font size="3"><b>July 15th</b></font><br>
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| - | <p align="justify">Construction of biobricks: The plasmid was purified according to the protocol.
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| - | Following purification, the plasmid was digested using two enzymes, XbaI (20U) and SpeI (10U). The mixture was incubated at 37ºC. 8 hours after incubation, more enzyme was added to ensure maximum digestion as well as Antartic phosphatase (25U). After one hour the mixture was run on a 1% agarose gel and the 2kb backbone was purified.<br><br>
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| - | Once we had the backbone where all three biobricks were going to be cloned, there was nothing left missing to start working on the biobricks.
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| - | All three biobricks were constructed by PCR from the pSVA13, a plasmid which was kindly obtained from Dr. Simon V. Avery at the University of Nottingham.
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| - | <font size="3"><b>July 14th</b></font><br>
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| - | <p align="justify">Construction of biobricks: Some colonies were picked from the transformation plate and made overnight cultures in liquid media.</p><br>
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| - | <font size="3"><b>July 13th</b></font><br>
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| - | <p align="justify">Construction of biobricks: We chose <a href="http://partsregistry.org/Part:pSB1A2">pSB1A2 </a> from iGEM spring 2009 DNA collection Kit Plate (well 11P). The crystallized DNA was ressuspended and transformed into competent E. coli cells.
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