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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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
:Picture taken by Jiaqi Wu

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