Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/03/02

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Examining Negative MSV Control Plates

The plates for the negative control sender were grown overnight in the 37°C incubator. The MSV plate had multiple colonies on it, as expected, and the water-only plate had no colonies, indicating that there was no source of contamination, as shown by the picture below:

Figure 1 - Negative MSV Control Plates
:Negative control sender plates

pSB1A3 Gel Extraction, Ligation, and Transformation

The gel slice of the cut pSB1A3 plasmid was gel extracted with a starting weight of 0.1 g. The gel extraction resulted in a DNA concentration of 4.14 ng/μL, which is close to nothing in terms of yield. Despite this, we went ahead with the ligation but had to use a larger total volume of 20 μL to account for the low DNA concentration. The ligation mixture contained the following reagents, as shown by the table below:

Table 1 - pSB1A3 Ligation Reaction Mixture
Reagent Volume (µL)
Vector (4.14 ng/μL) 12
Ligase 2
Ligase buffer 2
Water 4
Total 20

The ligated vector was then transformed into BL21 cells and plated. The plates were left to incubate at 37°C overnight.