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ColiGuard
Transformation of the BBa K112806 + BBa B0015 ligation
finO and finP with pGEM - confirmation
We performed a PCR for each miniprep samples we got, using a specific forward primer for our inserts (finO and finP) and a reverse primer that is specific for pGEM vector (primer M13, see pGEM manual). This will allow us to verify that we have the insert ligated into the plasmid and, further more, will allow us to check whether or not our inserts are indeed in the correct frame position.
According to the pictures, both finO and finP's PCR resulted in bands that reached the expected size in several samples.
Therefore, we concluded we sucessfully have finO and finP correctly ligated into pGEM vector. Next step is ligating them into biobrick's vector.
Marcelo
YeastGuard
New strategy: pGEM
The plate of E. coli transformed with the pDLD+Biofusion had only two colonies. We did the colony PCR to find correct constructions of the pDLD and Lysozyme in Biofusion. We found two fragments corresponding to pDLD, but the negative control amplified the same fragment too! We decided to do miniprep of both colonies and re-confirm it by digestion. We found one expected band of lysozyme. We did miniprep of this colony and chose an inespecific one as well.
We digested the Lysozyme+Biofusion vector and pJEN1+Biofusion vector with XbaI and PstI to recover the part fragment with one NotI site. The Lysozyme digestion worked so we purified the correct fragment, ligated it in biofusion again in order to recover the second NotI site. Unfortunately the pJEN1 digestion didn’t work.
pADH1+YFP
An electrophoresis was made with the product of the digestion performed yesterday. Then, we purified the bands corresponding to the size of the pADH1 in the biofusion vector and the YFP part.
So we made the ligation of the YFP part in the biofusion vector that contains the ADH1 promoter. Then we transformed this construction in E. coli that has grown O/N in the kanamicin media plate.
Wesley and Gleidson
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