Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/03/31

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03/31/2015

Sample Read# 260 280 260/280 ng/µL
1 0.007 0.004 1.882 6.81
1 0.012 0.006 1.9 12.003

Today Ryan helped me double digest more backbone with EcoRI and XbaI for the second ligation.
I PCR purified the reactions and eluted at 45uL with warm water.

After purification, I pipetted the following contents into PCR tubes:

  • note to self: use 2.0mL tubes next time if you are going straight into a transformation.
Insert Concentration (ng/uL) Volume of 62.5ng Insert (uL) Volume of 50ng backbone (uL) Water
AubI 29 2.16 4.17 10.67
BjaI 30 2.08 4.17 10.75
BraI 66 0.95 4.17 11.88
CerI 50 1.25 4.17 11.58
RpaI 30 2.08 4.17 10.75
EsaI 39 1.6 4.17 11.23
LuxI 45 1.39 4.17 11.44
LasI 36 1.74 4.17 11.09
SinI 47 1.33 4.17 11.5
Bb only 39 0 4.17 12.83


All backbone came from the 12ng/uL PCR-purified product.

2uL of T4 10x ligase buffer and 1uL of ligase were added to each reaction as well. The total volume is 20uL.
I incubated reactions at room temperature for 10 minutes. Then, I followed with a long transformation.

I pipetted all contents (20uL) of each tube into a new 2.0mL tube, then added 50uL of DH5αT cells to each. I also created a negative control with just cells and 20uL water. I had a total of 11 tubes. I allowed them to incubate on ice for 30 minutes, then heat shocked at 42°C for 35 seconds, and incubated on ice for another 2 minutes. Following this, I pipetted 750mL of SOC into each tube. Then I taped the tubes in an empty agar plate and let it shake in the shaker for 1 hour. After the hour, I spun the tubes down, removed 500uL of SOC, resuspended the fluids and pipetted the contents (~300uL) into plates that have been labeled and warmed. I used beads to mix the contents in the plate and then placed them upside down in the 37°C incubator overnight.