====Digestion of Biobricks BBa_E0840, BBa_I718017, BBa_J61000 and BBa_I718016====
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Today, we made miniprep ( Team:UNICAMP-Brazil/Protocols/Mini-Prep protocol 2) to selected colonies yesterday. After, we performed two PCR. The first with VF2 and VR primer and the second with Anticol F and Anticol R primer (designed by us) in order to prove the correct transformation. Lamentably, we didn’t get it. In addition we made also a digestion reaction with EcoRI and PstI. We didn’t see the band size expected. Definitively, the CeiB isn’t into plasmid. We decide to try searching. Otherwise we repeated the cell transformation with CeaB DNA, we made colony PCR but don’t see the right size band.
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. 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.
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 made miniprep ( Team:UNICAMP-Brazil/Protocols/Mini-Prep protocol 2) to selected colonies yesterday. After, we performed two PCR. The first with VF2 and VR primer and the second with Anticol F and Anticol R primer (designed by us) in order to prove the correct transformation. Lamentably, we didn’t get it. In addition we made also a digestion reaction with EcoRI and PstI. We didn’t see the band size expected. Definitively, the CeiB isn’t into plasmid. We decide to try searching. Otherwise we repeated the cell transformation with CeaB DNA, we made colony PCR but don’t see the right size band.
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