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 and confirm that our construction is right:
The expected size for the fragment excised with XbaI and SpeI from PY1 + RFP + BBa_J23100 and PY1 + RFP + BBa_B0015 is 1028 bp.
As we can observe in the gel photo, all plasmids presented bands compatible with the expected size for the digestion.
Now that we have confirmed that our construction in biobrick format is right they are ready to be sent to the registry! =)
We decided to send the PY1 promoter inserted in plasmid BBa_J23100. It is our biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K284008 BBa_K284008]
Fabi and Léo
PY Promoter - Mini-prep and restriction analysis
We found transforming colonies apparently. So, we made colony PCR in order to prove the correct transformation. We used the VF2 and VR primers. We run the agarose gel and???………Yess!!! we get!!!. We observed some samples with band size expected (~840bp)
We selected the samples 2-5-7-19-22. We will make miniprep tomorrow.
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.
"
