User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/24

AU CHEM-570 Lab Prep

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Objective Repeat BSA cloning into intein vectors with new, correct 3' primer. Bench work PCR purification Followed instructions for Promega Gel and PCR purification kit, using spin column method Purified experimental BSA PCR sample from yesterday Double-digest Tube sterile H2O 10X NEB2 100X BSA DNA NheI (10 kU/mL) SapI (2 kU/mL pTXB1His 14.5 μL 5 μL 0.5 μL 25 μL of 102 μg/mL 2.5μL 2.5μL pTXB1 14.5 μL 5 μL 0.5 μL 25 μL of 120 μg/mL 2.5μL 2.5μL BSA PCR 0 μL 5 μL 0.5 μL 39.5 μL from purified BSA PCR reaction in step 1 2.5μL 2.5μL →2h @ 37°C Gel purification Kathryn Muratore 17:28, 24 August 2011 (EDT):I forgot to phosphatase the vectors before loading the gel, so we will phosphatase after purification. 50μL pTXB1His double-digest from step 2 + 10μL 6X loading buffer 10μL 1KB ladder skip 50μL pTXB1 double-digest from step 2 + 10μL 6X loading buffer 50μL BSA PCR double-digest from step 2 + 10μL 6X loading buffer Spun down 4 x 500mL growths from yesterday (10 minutes at 3810rpm and 4°C) Resuspended pMAL-pIII in 100mL 30mM TrisCl 20% sucrose w/v pH 8.0 then placed in freezer @ -20°C°C Resuspended pMXB10 in 25 mL 20mM TrisCl 500mM NaCl pH 8.5 then quick froze with liquid nitrogen → freezer @ -80°C After gel purification, added 6μL 10x phosphatase buffer, 3μL dH2O, and 1μL phosphatase to the 50mL the two plasmids were eluted into. Heat inactivated phosphatase with 5 minutes at 65°C Tube sterile H2O 10X ligase buffer BSA plasmid T4 DNA ligase pTXB1 + BSA 13μL 5μL 20μL 10μL 2μL pTXB1 (-) 33μL 5μL 0μL 10μL 2μL pTXB1.His + BSA 13μL 5μL 20μL 10μL 2μL pTXB1.His (-) 33μL 5μL 0μL 10μL 2μL Results GEL