Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/05/22

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PCR clean up of EsaI PCR and mCh Sender Intermediate for golden gate. Forgot to do DpnI digest of mCh (don't need to for EsaI because the PCR template is kan resistance).

Sample 260 280 260/280 ng/µL
mCh Sender int gg 0.055 0.031 1.774 55.337
EsaI 0.046 0.026 1.776 45.692



picked all the colonies on the LuxR, RhlR, EsaR golden gate receivers with the 10:1 ratios and streaked them on a new plate. dotted 8ul of sender cells in the center of the plate. put at 37°C at 11am.

Going to run golden gate anyway. Backbone plate should have the same number of PCR template transformants as any of the ligation plates.
200fmol of insert, 20fmol backbone

Reaction Rec Bb LuxI BsmBI T4 Ligase T4 Ligase buffer H2O
Bb ctrl 0.77 0 0.5 1.25 2 15.48
RhlI 0.77 3.2 0.5 1.25 2 12.28
LasI 0.77 2.8 0.5 1.25 2 12.68
EsaI 0.77 1.9 0.5 1.25 2 13.58



Set up PCR of LuxI with P131 and P132 AT 50°C ext time 1 min because it failed the other day with an annealing temp of 50.