*<p style=”text-align:justify;”>We got transforming cell. We selected the white colonies and we made PCR in order to prove the correct transformation. For CeaB, we performed the reaction with pGEM M13 primer forward and ColR primer reverse meanwhile for CeiB, we used pGEM M13 primer forward and anticol R primer reverse. We run the agarose gel and this time we confirmed one good transformation. The CeiB was correctly inserted in the pGEM vector. Unfortunately, the CeaB colicin was not confirmed. For CeiB, we expected and found ~360 bp band size. </p>
Another Miniprep, another digestion, another ligation and once again, another transformation
Transformation of the BBa K112806 + BBa B0015 ligation
We did the miniprep of BBa B0015 and BBa K112806 according to Protocol 2
With the protduct of the miniprep we made the digestion of BBa B0015 with EcoRI and XbaI and with EcoRI and SpeI to BBa K112806. After 3 hours of digestion we purified BBa K112806 from agarose gel and BBa B0015 directly from the digestion reaction.
Using the purified digestions we did the ligation, Protocol 11, of BBa B0015 with BBa K112806 and to test the digestion of BBa B0015 we did a ligation only without BBa K112806, if colonies grow with this ligation it means BBa B0015 is recircularizing. We staterd to quantify our samples with the new lab's espectrophotometer.
We transformed E. coli with the ligation, we hope this time it works!!
Ane and Marcos
PY Promoter - Mini-prep
Today we performed mini-preps (Protocol 2) to extract the plasmids from the selected colonies inoculated yesterday.
Fabi and Léo
CeiB transforming in pGEM
We got transforming cell. We selected the white colonies and we made PCR in order to prove the correct transformation. For CeaB, we performed the reaction with pGEM M13 primer forward and ColR primer reverse meanwhile for CeiB, we used pGEM M13 primer forward and anticol R primer reverse. We run the agarose gel and this time we confirmed one good transformation. The CeiB was correctly inserted in the pGEM vector. Unfortunately, the CeaB colicin was not confirmed. For CeiB, we expected and found ~360 bp band size.
Luige & Ane
YeastGuard
New strategy: pGEM
We need more parts!! We performed PCR to obtain the following parts: End and JENorf again.
We inoculated JENorf-pGEM and Jen1promoter-pGEM transformed colonies in liquid media to perform the screening tomorrow.
Terminator
We did PCR from the DNA ressuspended from the kit plate corresponding to the "Terminator" biobrick and from the DNA extracted form Kluyveyromices lactis. We found the JENorf fragment, unfortunately we didn't find the terminator expected fragment.
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