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

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

1. 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
2. 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
3. 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
4. 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
5. 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