Team:Freiburg/Notebook/16 July

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red light receptor

Miniprep

Investigators: Jakob

RZ Plasmid Miniprep

Name: Jakob	Date: 16.07.2011
Continue from Experiment: -14.07.2011 -Ligation and transformation of ho1+terminator and PcyA+terminator
Project Name: Red light receptor

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:

• Parts: BBa_I15008 + B1006 and BBa_I1009 + B1006

• DNA concentration:

	DNA ng/	260/280	260/230
L22a(ho1+term)	90	1,9	1,99
L22b(ho1+term)	99,3	1,85	1,92
L23a(PcyA+term)	45,6	1,96	2,42
L23b(PcyA+term)	58,7	2,0	2,58

• Labeled: l22a, l22b, l23a, l23b Blue rack -> freezer