User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/17

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Objective

  • Run gel with test PCR reactions with differing reagents
  • Glycerol stock overnight growths: pMAL-pIII and pMXB10 in DH10B and ER2566
  • Optional miniprep of DH10B growths
  • Innoculate 2 x 500mL 2xYT media (+ 500μL 100mg/mL) with 10mL of 50mL overnight growths in ER2566 (grow, shaking at 37°C)
    • Induce expression when OD @600nm reaches 0.6 (about 2hours)
    • Grow overnight, shaking at 37°C

Bench work

  1. Made glycerol stocks of the overnight growths from last night
    • pMAL-pIII in DH10B + Amp → tube #112
    • pMXB10 in DH10B + Amp → tube #113
    • pMAL-pIII in ER2566 + Amp → #114
    • pMXB10 in ER2566 + Amp → #115
  2. Miniprepped DH10B samples of pMXB10 and pMAL-pIII (...concentrations not checked, run on gel)
  3. DNA gel (1% agarose):
    • Lane 1: Ladder (10μL)
    • Lane 2: ----
    • Lane 3: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer A and Pfu Turbo polymerase 1 8/16
    • Lane 4: ----
    • Lane 5: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer A and Pfu Turbo polymerase 2 8/16
    • Lane 6: ----
    • Lane 7: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer B and Pfu Turbo polymerase 1 8/16
    • Lane 8: ----
    • Lane 9: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer B and Pfu Turbo polymerase 2 8/16
    • Lane 10: ----
    • Lane 11: pMXB10 miniprep from today, 8/17
    • Lane 12: ----
    • Lane 13: pMAL-pIII miniprep from today, 8/17
  4. Innoculated 2 x 500mL of 2xYT media (+ 500μL 100mg/mL ampicillin) with 10mL of the 50mL ER2556 growths from last night (pMAL-pIII and pMXB10)
    • After a little under 2 hours, checked the OD @600nm and they were all overgrown by a good bit:
      • pMXB10 #1 = 0.764
      • pMXB10 #2 = 0.755
      • pMAL-pIII #1 = 0.663
      • pMAL-pIII #2 = 0.657
    • Immediately induced expression with 500μL 1M IPTG in each flask
      • Let grow, shaking overnight @37°C
  5. Made buffers for protein purification
    • 1M TrisCl pH 8.0, 100mL
      • 12.114g, used HCl to balance to pH 8.0; filtered through 0.45μm membrane
    • 30mM TrisCl pH 8.0 with 20% w/v sucrose, 200mL
      • 6mL of 1M TrisCl stock, 40g of sucrose, balanced pH with HCl; filtered through 0.45μm membrane


Results

Gel

DNAgel 8.17.2011 testPCR labeled.jpg