Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/03/31
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Today Ryan helped me double digest more backbone with EcoRI and XbaI for the second ligation.
After purification, I pipetted the following contents into PCR tubes:
2uL of T4 10x ligase buffer and 1uL of ligase were added to each reaction as well. The total volume is 20uL.
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.