User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/16

AU CHEM-570 Lab Prep

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Objective Run QuikChange on pMXB10 from yesterday(by Dr. Muratore) on gel Check transformations on plates Prepare for expression of pMAL-pIII and pMXB10/start cultures from colonies on plates Bench work Ran 1.0% agarose DNA gel with 2-stage QuikChange of pMXB10 for +His tag from yesterday(by Dr. Muratore) Lane 1: Ladder Lane 2: ---- Lane 3: ---- Lane 4: pMXB10 QC +His 8/15 Lane 5: ---- Lane 6: ---- Lane 7: pMXB10 QC Ctrl 8/15 Due to perceived failure of QuikChange from yesterday, performed a test PCR with pCMV-SPORT6+BSA to amplify BSA gene Vary Pfu Turbo buffer and Pfu Turbo polymerase in PCR reaction: Buffer A + Polymerase 1 == 'A1' Buffer A + Polymerase 2 == 'A2' Buffer B + Polymerase 1 == 'B1' Buffer B + Polymerase 2 == 'B2' Follow same protocol as 5/26 Took 1 colony from each of pMAL-pIII and pMXB10 in DH10B and put into 10mL of LB medium with 10μL of 100mg/mL ampicillin Grew O/N at 37°C Took 1 colony from each of pMAL-PIII and pMXB10 in ER2566 and put into 50mL of LB medium with 50μL of 100mg/mL ampicillin Grew O/N at 37°C Replated the four lowest-yielding transformations: pMAL-pIII and pMXB10 in ER2566 BamHI-digested pKK223-3 ligations of 10μL and 15μL in DH10B Results Gel Did not see what we wanted on gel -- suspect issue with reagents