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 Buffer 2.0 BbsI 1.0 Roche SAP 1.0 Water 14.5 Total 20 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 Buffer 3 EcoRI 1 SpeI 1 Water 13 Total 30 Incubate at 37°C for 10-20 minutes PCR purify PCR pExp and pLas Reagent Volume (uL) F Primer 1 R Primer 1 DNA Template 1 Taq 2x MM 25 Water 22 Total 50 Thermocycler: Temp (°C) Length (s) 95 30 95 15 60-70 30 72 15 72 5m 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.