IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/18
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Week 1 Summary (6/18/2010)
Aim: To biobrick agrobacteria vectors
To create the vector backbones we digested the E and R plasmids with HindIII and SpeI, and the O plasmids with SacII and SpeI. We ran the digested backbones on a gel and purified using the Qiaquick Gel Extraction kit. IMAGE HERE
To create the vector insert for the E plasmids, we designed primers such that we could amplify the promotor sequence present in the original plasmid and attach it to the biobrick multiple cloning site, bracketed with the appropriate restriction sites such that we could insert it into our digested backbone. The forward primer started with the HindIII restriction site to match the backbone, then included the biobrick cloning site, then the beginning of the promotor sequence to be amplified. The reverse primer started with the NheI restriction site (so that when ligated to the SpeI site in the backbone, a scar site would be created), then the end of the promotor sequence to be amplified.
We then ran a PCR reaction on the plasmids with these primers to obtain the vector insert, and digested with HindIII and NheI to prepare the inserts for insertion into the backbone
We plan to use annealed oligos to create the inserts for the O1 and O2 vectors.
We ligated the inserts with the backbones using T4 ligase. We transformed into NEB Turbo Competent E. coli cells and plated on LB and Kanamycin (50ug/ml). We picked colonies from each plate and made cultures.
LacI+NLS primers arrived in mail
spun at 14,000 rpm for 10 minutes diluted stocks to 100 μM
LacIn.BB.Rev (1.7nM) diluted with 17μL DH2O LacIn.BB.Fwd (1.5nM) diluted with 1011μL DH2O NLS.BB.Rev (4.2nM) diluted with 42μL DH2O LacIn.BB.Fwd (101.1nM) diluted with 15μL DH2O