User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/23

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Objective

  • Expression of MBP in ER2566
  • Redo BSA PCR from pCMV-SPORT6+BSA with correct 3' primers
  • Transform QuikChange product from 8/19

Bench work

  1. Added 500μL 100mg/mL ampicillin to 4 x 500mL 2xYT media in 2800mL Fernbach flask, then added 2.5mL of 40% glucose to each flask as well
    • Innoculate two flasks each with 10mL of pMAL-pIII and pMXB10 in ER2566 50mL growths
    • Grew 2 hours, then checked OD @600nm (looking for 0.600):
      • pMAL-pIII #1 OD = 0.677
      • pMAL-pIII #2 OD = 0.646
      • pMXB10 #1 OD = 0.622
      • pMXB10 #2 OD = 0.639
    • Induced expression with 500μL 1M IPTG, grew O/N @ 37°C
  2. Followed standard Transformation protocol with pMXB10 QuikChange product from Friday
  3. 3' SapI primer was incorrect, so PCR was redone today with correct primer just come in.


Results

  • No Colonies on plates from this day