Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/06/28

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06/28/2016

  • Digest LasR_MRV with BbsI
Reagent Volume (uL)
DNA (500ng)1.5
10x Green FD Buffer2.0
BbsI1.0
Roche SAP1.0
Water14.5
Total20
Incubate at 37°C for 10-20 minutes
Heat inactivate rSAP at 75°C for 2 minutes
Heat inactivate BbsI at 65°C for 20 minutes
  • Digest pCer and pBta3R
Reagent Volume (uL)
DNA (500ng)12
10x Green FD Buffer3
EcoRI 1
SpeI 1
Water13
Total30
Incubate at 37°C for 10-20 minutes
PCR purify


  • PCR pExp and pLas
Reagent Volume (uL)
F Primer1
R Primer1
DNA Template1
Taq 2x MM25
Water22
Total50
Thermocycler:
Temp (°C) Length (s)
9530
9515
60-7030
7215
725m
Extension for 15 minutes because of small size. Gradient of 60-70°C because pExp needs a higher annealing temperature to increase specificity; pLas uses 60°C to increase yield because it's plasmid is simple and does not contain similar binding sites. pExp was annealed at 66-70°C.



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