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

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