Team:EPF-Lausanne/Notebook/June2011

From 2011.igem.org

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(Gradient PCR)
(Gibson Assembly)
 
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RFP (820 bp) : 28.8 ng/microL
RFP (820 bp) : 28.8 ng/microL
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J6 (2334 bp) : 26 ng/microL
J6 (2334 bp) : 26 ng/microL
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'''Instead of following the instructions on the wiki by Nikolaus Obholzer which has the wrong formula for finding the equal molecular ratio''', we determined that the ratio ought to be 80/233 since the length of the RFP fragment is 820 bp and the length of the J6 plasmid is 2334. 
 
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'''Instead of following the instructions on the wiki by Nikolaus Obholzer which has the wrong formula for finding the equal molecular ratio''', we determined that the ratio ought to be 80/233 since the length of the RFP fragment is 820 bp and the length of the J6 plasmid is 2334. To make the Gibson mix, we used 2.6 microliters (80 ng % 28.8 ng/microL) of the RFP segment and 9 microliters of the plasmid (230 ng % 26 ng/microL). The assembly was done with a sample of 5 microliters taken from the 11.6 microliter mix.
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We then did a transformation with these Gibson-ed plasmids and plated the cells on ampicillin plates and put them in the incubator overnight.
==  Gradient PCR  ==
==  Gradient PCR  ==
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In a last attempt to try to amplify the P23019 plasmid backbone (the last piece needed to start a Gibson assembly of the TetR plasmid), Vincent tried a gradient PCR in which separate samples of the plasmid would be run at temperatures between 40 and 50 C (40, 42, 44, 46, and 48). The results showed, once again, that amplification was beyond reach.  
In a last attempt to try to amplify the P23019 plasmid backbone (the last piece needed to start a Gibson assembly of the TetR plasmid), Vincent tried a gradient PCR in which separate samples of the plasmid would be run at temperatures between 40 and 50 C (40, 42, 44, 46, and 48). The results showed, once again, that amplification was beyond reach.  
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[[File:EPFL-29-06-p23019fail.jpg]]
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[[File:EPFL-29-06-p23019fail.jpg|300px|GFP-tetR and cy5 fluorescence]]
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{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 16:51, 9 July 2011