- 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
- 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
- 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
- Replated the four lowest-yielding transformations:
- pMAL-pIII and pMXB10 in ER2566
- BamHI-digested pKK223-3 ligations of 10μL and 15μL in DH10B
- Did not see what we wanted on gel -- suspect issue with reagents