Team:University of Chicago/Notebook

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Contents

Lab Calendar

Click to see what lab work was done each day!


Individual Lab Notebook Pages

Remember: If you did it, record it

Resources

[http://www.freewebs.com/genehackers/ Team Website (External Link)]

Recipes

Protocols

Glycerol Stocks
Materials
  • 40% glycerol solution
  • Cryogenic vials
Method
  1. Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
  2. Add 1 ml sample from the culture of bacteria to be stored.
  3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
    • Alternatively, pipet to mix.
  4. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
  5. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
  6. Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.
Notes

While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase. Prof. Schonbaum's version

  1. Grow a 3 ml culture overnight
  2. Add 200 µl 100% glycerol to 1 ml cells in a cryotube (final concentration should be 15-20% glycerol different concentrations from different protocols
  3. Mix by vortexing and let sit for a little bit. Then put the tubes in the -80°C freezer.

DNA prep

Zyppy mini-prep

The following procedure is performed at room temperature. Ensure that RNase A has been added to the Neutralization Buffer, ethanol has been added to the Zyppy™ Wash Buffer, and that the 7X Lysis Buffer has not precipitated during shipping (to completely resuspend the buffer, incubate the bottle at 30 – 37 ºC for 30 minutes and mix by inversion).

  1. Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube
    • The Zyppy™ Plasmid Miniprep Kit may also be used with the classical centrifuge- based procedure for processing up to 3 ml of bacterial culture. The procedure should be modified as follows: 1A) Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed. 1B) Discard the supernatant. 1C) Repeat steps 1A and 1B as needed. 1D) Add 600 μl of TE or water to the bacterial cell pellet and resuspend completely.
  2. Add 100 µl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.
  3. Add 350 µl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete Proxy-Connection: keep-alive

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utralization.

  1. Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.
  2. Transfer the supernatant (~900 µl) into the provided Zymo-Spin™ IIN column.Avoid disturbing the cell debris pellet.
  3. Place the column into a Collection Tube and centrifuge for 15 seconds.
  4. Discard the flow-through and place the column back into the same Collection Tube.
  5. Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube.
  6. Add 400 µl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds.
  7. Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 µl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature.
  8. Centrifuge for 15 seconds to elute the plasmid DNA.

Notes

  1. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, we recommend working with groups of ten or less at a time. Continue with the next set of ten samples after the first set has been

neutralized and mixed thoroughly.

  1. Ensure that RNase A has been added to the Neutralization Buffer and that ethanol has been added to the concentrated Zyppy™ Wash Buffer.
  2. The ZyppProxy-Connection: keep-alive

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�� Elution Buffer contains 10 mM Tris-HCl, pH 8.5 and 0.1 mM EDTA. If required, pure water (neutral pH) can also be used to elute the DNA.

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