User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/15

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Current revision

AU CHEM-570 Lab Prep Main project page
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Contents

Benchwork

Plan

  • Miniprep pTXB1His and send for sequencing
  • Start more cultures of new colonies for more minipreps
  • Don't forget to miniprep pTXB1 (unmodified) as well
  • Streak expression strain (ER2566) on LB so competent cells and a glycerol stock can be made

Experiments

  1. Glycerol stock
    • 750μL pTXB1His/DH10B QC colony #1 culture from yesterday + 250 μL 60% glycerol (sterile) → tube #79
    • → -80°C
  2. Miniprep of pTXB1His
    1. follow Miniprep vacuum protocol for pTXB1His/DH10B QC colony #1 culture from yesterday
  3. Quantify [pTXB1His miniprep #1]
    1. 95μL H2O
    2. + 5μL miniprepped DNA from step 2
    • → measure absorbance at 260 nm and 280 nm
  4. DNA sequencing
  5. Miniprep cultures of pTXB1His and pTXB1 in DH10B
    1. 10mL LB + 10μL 100 mg/mL Amp + pTXB1His/DH10B QC colony #2 (see Results below)
    2. 10mL LB + 10μL 100 mg/mL Amp + pTXB1His/DH10B QC colony #3 (see Results below)
    3. 10mL LB + 10μL 100 mg/mL Amp + pTXB1/DH10B colony (from last week)
      • → 37°C O/N w/ shaking

Results

Transformations

  • Four plates yesterday:
    • 200μL experimental transformation from yesterday: 1 colony
      • → pTXB1His/DH10B QC colony #2
    • 200μL control transformation from yesterday: 0
    • 800μL experimental transformation re-plated from 2 days ago: 1 colony
      • → pTXB1His/DH10B QC colony #3
    • 800μL control transformation re-plated from 2 days ago: 0

Miniprep

[pTXB1His\#1]=(0.114AU+0.012AU)\times50\frac{\frac{\mu g}{mL}}{AU}\times\frac{100\mu L}{5\mu L}=102\frac{\mu g}{mL}

\frac{A_{260}}{A_{280}}=\frac{0.114AU+0.012AU}{0.067AU+0.011AU}=1.82

Sequencing

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