Lidstrom:Protocols

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(E. Coli)
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==E. Coli==
==E. Coli==
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]]
*[[Lidstrom:Competent Cell Preparation|Competent Cell Preparation (in progress)]]
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==Electrocompetent Cells==
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*You can make your own electro-competent cells for electroporation.
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**From Amada's past mentor in undergrad:
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***Grow the cells overnight
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***Inoculate from this the next morning: generally use 200 uL/50 mL
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***Let grow for about 3-4 h (OD 0.4-0.6 or so),  then centrifuge and wash 3 times with 10% glycerol (everything on ice).
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***Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
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****Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care.  See [[Electrocompetent_Cells| this page]].  Would allow you to calculate efficiency.

Revision as of 20:56, 2 July 2012

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Contents

General Lab:

Cell Culture:

DNA/RNA:

DNA

RNA

E. Coli

Electrocompetent Cells

  • You can make your own electro-competent cells for electroporation.
    • From Amada's past mentor in undergrad:
      • Grow the cells overnight
      • Inoculate from this the next morning: generally use 200 uL/50 mL
      • Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).
      • Resuspend the pellet in a small volume (Example, if I do a 5 ml culture I try to resuspend the final pellet in no more than 0.2 or 0.3 ml of 10% glycerol). Then split in 1.5 ml centrifuge tubes (0.1 ml in each), freeze on dry ice-isopropanol and store at -80C.
        • Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page. Would allow you to calculate efficiency.
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