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Team:Groningen/Template/MODULE/Notebook/secretion/week6
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1 – 5 September
goal: obtain ssUSP45DspB in pSB1C3 biobrick and ssUSP45Aiia in pSB1C3 biobrick.
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.
2 series of PCR ran
Amplification of gblock in 50µl reaction
Amplification of product: 10*50 µl reaction
The products were ligated into 70 ng of pSB1C3 and transformed into E. coli .
Another PCR was done with the amplificated PCR product of ssUSPdspB, these products were
The signal sequence of USP45
The gblock without HIS-tag.
These products were also ligated into pSB1C3 and transformed into 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 presence of ssUSP45aiiA, then it got ligated into pSB1C3 and transformed Into E. coli as well.
Results of the transformed E. coli gave a yield of 40% of possible clones.
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.
