Part:BBa_J100182:Experience

Our first step after acquiring the synthesized oligos we boiled them with 4 microliters of 10x annealing buffer (IM NaCL, 100mM Tris-HCl, pH 7.4), 1 microliter of the top oligo, 1 microliter of the bottom oligo, and 14 microliters of water for a total concentration of 20 microliters. Then, we incubated them for about 20 hours. Then we diluted the oligo 100 fold using 1 microliter of the oligo combined with 99 microliters of water, we added 1 microliter of these diluted oligos to the 9microliters of already made GGA mix (contained the plasmid J1119137, Bsa1 and ligase) for our experimental ligation and for our negative control we added 1 microliter of water instead of the oligos. Then we put them in the thermocycler for 20 cycles at 37 degrees C for one minute, then at 16 degrees C for one minute, I cycle of 37 degrees C for 15 minutes, then at 22 degrees C to hold temperature. Then we pipetted the transformed oligos onto the plates that contained LB+ amp, making sure to spread the oligo solution.

The next day, our instructor checked our plates and identified that every groups' plates were red despite the negative control which should have been green. The plasmid given to the oligos was incorrectly labeled so the results were skewed, but the correct plasmid was then administered to the oligos and the results came out as hypothesized.

Colonies (2) were then picked for extraction from each of the plates and added to 100 microliters of 2M solution of ammonium chloride to one of each of the separate colonies picked. We pipetted 8 replicates of each colony tested (both with and without ammonium chloride) into a well plate that we then used a synergy machine to measure how much RFP each culture of cells was producing. The synergy machine gave us data that we then averaged to find the average fluorescence per cell. The error bars are standard deviation of that average.

Applications of BBa_J100182

Used the previous reference numbers, the + indicates treatment with NH4Cl and - was treated in the absence of NH4Cl

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