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
10.0070.0041.8826.81
10.0120.0061.912.003

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
AubI292.164.1710.67
BjaI302.084.1710.75
BraI660.954.1711.88
CerI501.254.1711.58
RpaI302.084.1710.75
EsaI391.64.1711.23
LuxI451.394.1711.44
LasI361.744.1711.09
SinI471.334.1711.5
Bb only 3904.1712.83


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.

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