Team:UNICAMP-Brazil/Notebooks/October 13

ColiGuard

• Today we performed mini-preps (Protocol 2) to extract the plasmids from the selected colonies inoculated yesterday.

• After plasmid extraction we digested them with XbaI and PstI to analyze the sizes of the resulting bands on the agarose gel:

Fabi and Léo

PY Promoter - Mini-prep and restriction analysis

YeastGuard

New strategy: pGEM

• The final confirmation of the lysozyme biobrick was done by digestion of the three plasmids chosen yesterday with EcoRI and PstI. The digestion confirmed two lysozyme biobricks (~600bp of the part and ~3000bp of the vector).

• We did miniprep of the promoters’ biobricks and digested the plasmids with EcoRI and PstI to confirm the correct insertion. The digestion of pJEN1 confirmed 1 positive colony (~1000bp) and the pDLD digestion confirmed 2 colonies (~500bp).

• We digested the correct biobricks with SpeI and PstI in order to open the vectors and connect the parts. The digestion showed expected fragments (pDLD: 3729bp and pJEN1: 4264bp).

• We purified the fragments from the agarose gel and connected them to lysozyme and to the reporter gene YFP, both digested with XbaI and PstI. Then we transformed these ligations in competent E. coli and plated in LB+Amp media.

Raíssa and Taís