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Team:TU-Delft/Notebook/2013/07/23/
From 2013.igem.org
Notebook
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23rd of July
Lab work
1. The Gel extraction of pTET:cI and AgrAC:pP2:GFP were carried out which were ligated with TT and pBAD respectively.
2. These were then transformed to TOP 10 cells, plated on Agar, grown in culture media, and DNA was extracted from it. Today gel electrophoresis was carried out on it.
3. Transformation of AIP sensor in BL21 cells was done and glycerol stocks were made. The plate looked good.
4. The oligopeptides need to go into pSB1C3 backbone, for which we restriction digest the AIP receiver with XbaI + PstI. Gel electrophoresis showed good results. Gel extraction was done to retrieve the backbone pSB1C3.
5. Sent samples of AIP:GFP fw/rv, pTET:cI fw/rv, AIP:GFP:pBAD fw/rv, pTET:cI:TT fw/rv for sequencing.
6. The oligoprimers were hybridized by adding forward and reverse sequences and running in PCR at 98 Degree to 75 Degree for 30 seconds each.
7. The previous quick change of SUMO and Ulp1 failed. So quick change was again carried out at 49 degree annealing temperature. Transformation was done on TOP 10 Cells and plate on Agar plates.
8. The primers, peptides(Maximin H5, Signiferin and Magainin) and pSB1C3 were PCRed and transformed into DH5Alpha cells.
