This morning we found some E. coli colonies that were transformed yesterday! We performed PCR with 10 colonies from each biobrick (pJEN1, pDLD and Lysozyme) with the vector's primers (VR and VF).
The figure shows that unfortunately none of the colonies tested had the insert into the vector even with its dephosphorylation. =( The size of the amplicons correspond to the vector's fragment without any insert (+/- 1400bp).
We also decided to try to do a miniprep using the rest of the transformed E. coli that was not plated in LB+AMP media. So today we made the inoculum.
Thinking about another strategy to avoid recircularization and antisense insertion...
Raíssa and Taís
Customizing the PCR
Electrophoresis of the Jen1 (ORF) product PCR to purify the band and proceed a new JEN1 (ORF) PCR.
Wesley and Gleidson
ColiGuard
Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016
After confirming the miniprep procedure, we digested the cited biobricks with the correct enzymes in order to make possible the construction of our necessary devices. Therefore, we had to:
- BBa_E0840: isolate the part in order to ligate it in the downstream position of another biobrick (digested with XpeI and PstI)
- BBa_I718017: open the biobrick in the downstream position of it's part (digested with SpeI and PstI)
- BBa_J61000: isolate the part in order to insert it in the upstream position of another biobrick (digested with EcoRI and SpeI)
- BBa_I718016: open the biobrick in the upstream position of it's part (digested with EcoRI and XbaI)
Digestion lasted 3 hours on all cases.
Marcelo
Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016
Today, we performed miniprep (Protocol 2) of the selected colonies yesterday. After that, we performed two PCR. The first reaction with VF2 and VR primers and the second reaction with Anticol F and Anticol R primers (designed by us) in order to comprove the correct transformation. Lamentably, we didn’t get it. In addition we performed a digestion reaction with EcoRI and PstI. We didn’t see the expected band size. Definitively, the CeiB isn’t into the plasmid. We tried a search. Otherwise, we repeated the cell transformation with CeaB DNA and colony PCR but didn’t see the right size band.
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