Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/02/17

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02/17/2015

Ligation:
Today I ligated the insert and backbone prepared last week via PCR purification.
3 ligations and 2 backbone-only controls were prepared as follows:

Ratio (in:bb) Insert (μL) Backbone (μL) DNA dilution buffer (μL)
1:3 2.6* 1.59 5.81
1:1 3.9* 0.79 5.31
3:1 1.19 0.79 8.02
Bb only (100ng) 0 1.59 8.41
Bb only (50ng) 0 0.79 9.21


Insert was diluted x10 (1μL insert per 9μL water)*
Calculations were done based on the concentrations of the backbone and insert; both were 63ng/μL.

10μL of DNA dilution buffer was then added to each tube, followed by 2μL(?) of ligation buffer and then finally, 1μL of ligase.
The ligation was set to incubate at room temperature for 5-15 minutes.

Transformation
I added 50μL of DH5αT cells to each of my reaction, including an additional positive and negative control. Then, I taped these inside a plate and put the plate in the shaker to shake for 1 hour.

Rene: Pippet the reactions into an AMP agar plate, pour in ~8 glass beads and shake. Then store in 37C incubator overnight. Transfer to 4C fridge.