Team:Groningen/Template/MODULE/Notebook/secretion/week6

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September 1 - September 5
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<b>goal:</b> obtain ssUSP45DspB in pSB1C3 biobrick and ssUSP45Aiia in pSB1C3 biobrick.
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Obtaining ssUSP45DspB (<a href="http://parts.igem.org/Part: BBa_K1365111">BBa_K1365111</a>) as a BioBrick in the pSB1C3 vector and ssUSP45Aiia (<a href="http://parts.igem.org/Part:BBa_K1365169">BBa_K1365169</a>) as a BioBrick in pSB1C3 vector
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Because of the very low amount of gblocks given, a decision was made to do a PCR directly on the product itself, therefor multiplying it exponentially.
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2 series of PCR ran
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<div class="item">Amplification of gblock in 50µl reaction</div>
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<div class="item">Two PCR reactions directly on the products were performed</div>
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<div class="item">Amplification of product: 10*50 µl reaction</div>
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<div class="item">The products were ligated with pSB1C3, and transformed to <i>E. coli</i> DH5α</div>
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<div class="item">Another PCR was done with the amplificated PCR product of ssUSPdspB, one with the signal sequence of USP45, and one with the ssUSP45DspB without His-tag</div>
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<div class="item">These products were also ligated with pSB1C3, and transformed to <i>E. coli</i></div>
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<div class="item">Afterwards, Gibson assembly had been done with modified Aiia and the signal sequence of USP45 making ssUSP45aiiA, to enhance the chances of successfully ligating the Gibson product into pSB1C3, PCR was done on the final Gibson product</div>
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<div class="item">The PCR product was checked on gel, giving positive results for the ssUSP45aiiA, then it was ligated with pSB1C3, and transformed to <i>E. coli</i></div>
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<div class="item">Several colonies were visible on the plated transformation products</div>
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<div class="item">Colony PCR was done on them with the regular pSB1C3 test primers, which showed no results</div>
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<div class="item">The colonies were grown overnight, and the plasmids were isolated in the next week</div>
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The products were ligated into 70 ng of pSB1C3 and transformed into <i>E. coli </i>.
 
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Another PCR was done with the amplificated PCR product of <i>ssUSPdspB</i>, these products were
 
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<div class="item">The signal sequence of USP45</div>
 
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<div class="item">The gblock without HIS-tag.</div>
 
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These products were also ligated into pSB1C3 and transformed into <i>E. coli</i>.
 
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Afterwards, Gibson assembly had been done with modified Aiia and the signal sequence of USP45 making ssUSP45aiiA. to enhance the chances of successfully ligating the Gibson product into pSB1C3, PCR was done on the final gibson product.
 
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The PCR product was checked on gel, giving positive results for presence of ssUSP45aiiA, then it got ligated into pSB1C3 and transformed Into <i>E. coli</i> as well.
 
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Results of the transformed <i>E. coli </i>gave a yield of 40% of possible clones.
 
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Colony PCR was done on them with the regular pSB1C3 test primers, but no product was seen. Therefor 40 possible clones were grown O/N and mini-prepped the next week.
 
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Latest revision as of 23:55, 17 October 2014

September 1 - September 5
 
Obtaining ssUSP45DspB (BBa_K1365111) as a BioBrick in the pSB1C3 vector and ssUSP45Aiia (BBa_K1365169) as a BioBrick in pSB1C3 vector
 
Two PCR reactions directly on the products were performed
The products were ligated with pSB1C3, and transformed to E. coli DH5α
Another PCR was done with the amplificated PCR product of ssUSPdspB, one with the signal sequence of USP45, and one with the ssUSP45DspB without His-tag
These products were also ligated with pSB1C3, and transformed to E. coli
Afterwards, Gibson assembly had been done with modified Aiia and the signal sequence of USP45 making ssUSP45aiiA, to enhance the chances of successfully ligating the Gibson product into pSB1C3, PCR was done on the final Gibson product
The PCR product was checked on gel, giving positive results for the ssUSP45aiiA, then it was ligated with pSB1C3, and transformed to E. coli
Several colonies were visible on the plated transformation products
Colony PCR was done on them with the regular pSB1C3 test primers, which showed no results
The colonies were grown overnight, and the plasmids were isolated in the next week
 
 
 
 
 

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