User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/07

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Objective

  • Try electroporation
  • Re-ligate with different I:V

Bench work

  1. Glycerol stock of ER2566
    • 250μL 60% sterile glycerol + 750μL ER2566 culture from yesterday 80
  2. Electrocompetent DH10B
    t=0 OD600=0.08
    t~1.5h OD600~0.2
    t~2h OD600~0.6 → stop growth
    • incubate culture ~10' on ice w/ swirling
    • spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C Sorvall RC6+ and SH-3000 rotor
      • supernatent
        • discard
      • pellet
        • resuspend in 100 mL cold sterile H2O
        • spin in 2x50 mL falcon tube 20' @ 4500 RPM @ 4°C
          • supernatent
            • discard
          • pellet
            • resuspend in 45 mL cold sterile H2O (should be 50 mL, but not enough sterile water)
            • spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C
              • supernatent
                • discard
              • pellet
                • resuspend in 10 mL cold sterile 10% glycerol
                • spin in 15 mL falcon tube 10' @ 3800 RPM @ 4°C
                  • supernatent
                    • discard
                  • pellet
                    • resuspend in 200 μL cold sterile 10% glycerol
                    • aliquot into 5 x 50μL (*Kathryn Muratore 15:34, 8 July 2011 (EDT): I meant to freeze the 5th unused aliquot, but did not)
  3. Transformation by electroporation
    • Used David Carlini's Bio-Rad electroporator in Biology
    • 0.1 cm cuvettes
    1. 50μL DH10B + 20μL V(His)+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
    2. 50μL DH10B + 20μL V(His) neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
    3. 50μL DH10B + 20μL V+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
    4. 50μL DH10B + 20μL V neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
      • 1.5 kV, t = 3.6-4.0 ms
      • add 450μL SOC
      • → 1.5h @ 37°C w/ shaking (should be 45')
      • plate 100μL of 1:1 for all samples on LBAmp100
      • plate 100μL of 1:100 (diluted with SOC) for V(His)+I and V+I on LBAmp100
      • → 37°C O/N
  4. Re-plate transformants
    • spread remaining 450μL of NovaBlue transformations from yesterday
      • → 37°C O/N
  5. Ligation
    1. 8.5μL ddV(His) from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
    2. 8.5μL ddV from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
      • → 16°C O/N

Results

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