User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/15

From OpenWetWare

Jump to: navigation, search
AU CHEM-570 Lab Prep Main project page
Previous entry      Next entry

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

  1. QuikChange
    • Repeat June 13th's 2-stage, 5-step QuikChange exactly, but use newly-diluted pMXB10 instead of pTXB1.
    • extension cycle time = 8 minutes
  2. 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
Personal tools