Team:EPF-Lausanne/Notebook/June2011

From 2011.igem.org

(Difference between revisions)
(→Tuesday, June 28 2011)
(→Gibson Assembly)
 
(6 intermediate revisions not shown)
Line 206: Line 206:
*Primer 2: J61002-RFP-f
*Primer 2: J61002-RFP-f
*DNA template: Plate 1, 18 C  
*DNA template: Plate 1, 18 C  
 +
2) a J61002 plasmid backbone with a terminator (2334 bp)
2) a J61002 plasmid backbone with a terminator (2334 bp)
Line 211: Line 212:
*Primer 2: J61002-term-r
*Primer 2: J61002-term-r
*DNA template: Plate 1, 18 C
*DNA template: Plate 1, 18 C
 +
3) a constitutive promoter for TetR (725 bp)  
3) a constitutive promoter for TetR (725 bp)  
Line 222: Line 224:
*Primer 2: P23019-f
*Primer 2: P23019-f
*DNA template: Plate 4, 14E
*DNA template: Plate 4, 14E
 +
With a first PCR, we were able to amplify the pTet-RFP segment and the promoter for TetR, but neither of the backbones was amplified. The iProof PCR was done using 40 seconds of elongation time at 55 C.  
With a first PCR, we were able to amplify the pTet-RFP segment and the promoter for TetR, but neither of the backbones was amplified. The iProof PCR was done using 40 seconds of elongation time at 55 C.  
Line 254: Line 257:
This experiment confirms that wtTetR is expressed and correctly folded by SP6 expression system. The TF recognizes tetO1 consensus sequence while the tested random sequence (-)control has a significantly lower binding affinity to TetR.     
This experiment confirms that wtTetR is expressed and correctly folded by SP6 expression system. The TF recognizes tetO1 consensus sequence while the tested random sequence (-)control has a significantly lower binding affinity to TetR.     
-
On the Gibson assembly side of things, Vincent and Irina purified the PCR product of both the J61002 plasmid and the pTet-RFP construct using standard elution.
 
 +
== Gibson Assembly==
 +
 +
 +
On the Gibson assembly side of things, Vincent and Irina purified the PCR product of both the J61002 plasmid and the pTet-RFP construct using the standard PCR purification protocol. Then, using NanoDrop, they measured the concentrations of J61002 plasmid and of the RFP construct:
 +
 +
 +
RFP (820 bp) : 28.8 ng/microL
 +
 +
 +
J6 (2334 bp) : 26 ng/microL
 +
 +
 +
'''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.
 +
 +
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  ==
 +
 +
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.
 +
[[File:EPFL-29-06-p23019fail.jpg|300px|GFP-tetR and cy5 fluorescence]]
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 16:51, 9 July 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Notebook/June2011"