User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30

AU CHEM-570 Lab Prep

Main project page Previous entry      Next entry

Objective Continue trying to get BSA-intein construct cloned. Bench work Ligation Using new digested DNA Previous purification could have been problematic, but hopefully the yield is higher and there is less ethanol this time. Insert: gel-purified NheI/SapI digested BSA PCR from yesterday Vector(His): gel-purified NheI/SapI/Phosphatased pTXB1His1 from yesterday Vector: gel-purified NheI/SapI/Phosphatased pTXB1 from yesterday 8.5μL Insert + 8.5μL Vector(His) + 2μL buffer + 1μL T4 DNA Ligase 8.5μL Insert + 8.5μL Vector + 2μL buffer + 1μL T4 DNA Ligase 8.5μL Vector(His) + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control) 8.5μL Vector + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control) → 16°C O/N → store @ -20°C Transformation Tranform yesterday's O/N ligation into DH10B Follow standard transformation protocol except: 100μL DH10B + 5μL V(His)+I from yesterday 100μL DH10B + 5μL V+I from yesterday 100μL DH10B + 5μL V(His) neg ctrl from yesterday 100μL DH10B + 5μL V neg ctrl from yesterday heat shock time is 45s plate 100μL each Re-plate yesterday's NovaBlue transformation 450μL of each re-plated → 37°C O/N