Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/02/09

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02/09/2016 Examining pLas + LasR Ligation Plates Jiaqi performed ligations with pLas and LasR, and after overnight incubation at 37°C, the plates were as shown in the picture below: Figure 1 - pLas + LasR Plates : The plates exhibited the following distribution of colonies: pLas 1 (insert) / 2 (backbone) - Many colonies pLas 2/2 - ~15 colonies Both of the plates above had a similar number of colonies to the LasR Bb gel 2 plate, which indicates that most of the colonies grown only contained the backbone re-annealed to itself, instead of the backbone with the insert pLas 1/1 - ~10 colonies pLas 2/1 - 4 colonies These two plates above also showed the same pattern of colonization as the pLas plates with Bb 2 LasR Bb gel 1 - 4 colonies LasR Bb gel 2 - Many colonies Negative control - 1 colony Despite this single colony, it was determined that the other plates were not affected by contamination, as they had a substantial amount of colonies grow on them Generally speaking, there were many satellite colonies that grew around the larger, major colonies. Preparing gBlock Positive Control for Sequencing The general mixture for sequencing consists of... 1.6 µL of 10 µM primer 500 ng of DNA Up to 10 µL of water Total volume of 10 µL The mixtures containing the positive control were prepared as shown in the table below: Table 1 - gBlock Sequencing Mixtures Reagent Sequencing Mixture 1 2 3 4 5 Primer (µL) 1.6 1.6 1.6 1.6 1.6 DNA (µL) 3.8 4.3 4.4 5.7 5.1 Water (µL) 4.6 4.1 4.0 2.7 3.3 Total (µL) 10.0 10.0 10.0 10.0 10.0 The sequencing mixture numbers given in the table above correspond to the following plasmids: 1 = gB015 1 2 = gB015 2 3 = gB015 3 4 = gB015 gel 1 5 = gB015 gel 2