Objective
- Prepare for expression and purification of maltose binding protein (with and without intein) from pMAL-pIII and pMXB10
- Transform pMAL-pIII and pMXB10 into both DH10B and ER2566
- Transform pKK223-3 ligations from last week
Bench work
- QuikChange
- Repeat June 13th's 2-stage, 5-step QuikChange exactly, but use newly-diluted pMXB10 instead of pTXB1.
- extension cycle time = 8 minutes
- Transformations ( standard protocol):
- pMXB10 into DH10B and ER2566 competent cells (from 7/21/11 and 7/15, respectively)
- pMAL-pIII into DH10B and ER2566
- pKK223-3 BamHI-digested, ligated w/ 15μL in reaction solution ( 8/10) into DH10B
- pKK223-3 BamHI-digested, ligated w/ 10μL in reaction solution ( 8/10) into DH10B
Results
- Transformations (as reviewed on 8/15):
- 1 colony each on the pMXB10, pMAL-pIII in ER2566 plates at end of day
- 2 and 3 colonies (with satellites) on the pKK223-3 BamHI-digested ligation in DH10B plates at end of day
- Over a hundred colonies on pMAL-pIII in DH10B at end of day
- About 15-20 colonies on pMXB10 in DH10B plate at end of day
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AU CHEM-570 Lab Prep
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