Team:Freiburg/Notebook/18 August

• < home >
• < team >
  • → members
  • → attributions
  • → freiburg
• < project >
  • → description
  • → results
  • → modeling
  • → notebook
  • → sample data
• < human practices >
  • → oath
  • → ethics
  • → safety
• < gallery >
• < sponsors >
• < to the forums >

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

Miniprep

zymo Kit

Procedure:

The following procedures are carried out at a room temperature. All centrifugation steps should be performed between 11,000 – 16,000 x g

1.Centrifuge 0.5 – 5 ml of bacterial culture in a clear 1.5 ml tube at full speed for 15 – 20 seconds in a microcentrifuge. Discard supernatant.

2.Add 200 µl of P1 Buffer (Red) to the tube and resuspend pellet completely (i.e., by vortexing or pipetting).

3.Add 200 µl of P2 Buffer (Green) and mix by inverting the tube 2 – 4 times. Cells are completely lysed when the solution appears clear, purple and viscous. Proceed to the next step within 1-2 minutes.

4.Add 400 µl of P3 Buffer (Yellow) and mix gently but thoroughly. Do not vortex. The sample will turn yellow when the neutralization is complete. Allow the lysate to incubate at room temperature for 1-2 minutes.

5. Centrifuge sample(s) for 2 minutes.

6.Place a Zymo-Spin IIN column in a Collection Tube and transfer the supernatant from step 5into the Zymo-Spin IIN column. When pipetting the supernatant be careful not to disturb the green pellet to avoid transferring anz cellular debris to the column.

7.Centrifuge the Zymo-Spin IIN/Collection Tube assembly for 30 seconds.

8.Discard the flow-through in the Collection Tube, making sure the flow-through does not touch the bottom of the column. Return the Zymo-Spin IIN column to the Collection Tube

9.Add 200 µl of Endo-Wash-Buffer to the column and centrifuge for 30 seconds.

10. Add 400 µl of Plasmid Wash Buffer to the column. Centrifuge for 1 minute.

11. Transfer the column into a clean 1.5 ml microcentrifuge tube and then add 30 µl of DNA Elution Buffer to the column. Centrifuge for 30 seconds to elute the plasmid DNA.

Documentation:

Why are you doing this experiment? Name the parts which you extract.

ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too.

Name of the parts: GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2

Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.

How did you label your probes and where are they stored?

Testdigest

For one reaction you need: For Mastermix: Number of samples+2extra

10 μl total volume

Give 3 μl of DNA in an eppi and add 7μl of the mastermix.

Incubate for about 1h at 37°C.

Add 1 μl Loading dye buffer and load the gel.

Take a picture of the gel, print picture and label the lanes!