Notebook:Federico Castro M/2008/01/08

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Proposed Procedure

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• Project: Diffusible signal oscilator.
• Status: Pending for approval
• Issues: None

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I want to recover 4 biobricks in order to see their behavior. The biobricks are oscillators dependent on quorum sensing signals such as the one I want to assemble. I await for approval for this procedure.

Biobricks to be extracted

• <bbpart>BBa_I4203</bbpart> available at well 13K at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1AK3
• <bbpart>BBa_I4202</bbpart> available at well 13I at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1AK3
• <bbpart>BBa_I4201 </bbpart>available at well 13G at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1AK3
• <bbpart>BBa_I4200 </bbpart>available at well 13E at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1AK3

The following protocol is proposed for biobrick recovery from the iGEM page [1]

1. Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick™-standard part that you want
2. Add 15 uL of diH2O (deionized water)
3. Take 1uL DNA and transform into your desired competent cells, plate out on a plate with the correct antibiotic* and grow overnight. Your goal here is to obtain single colonies.

I intend to use the following protocol for transforming the biobricks into competent cells.

1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
3. Incubate on ice for 30 minutes
   • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
4. Incubate cells for 30 seconds at 42°C.
5. Incubate cells on ice for 2 min.
6. Add 4 volumes of room temperature SOC (not critical)
7. Incubate for 1 hour at 37°C on shaker.
   • Note: Can also save some time here by reducing incubation to ~45 min.
   • Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
9. Grow overnight at 37°C.

Before the procedure I would need to prepare solid plates with Kanamicyn (all of them have resistance to Kanamycin). It seems that the proper concentration of Kanamicyn is 50 μg/mL.[2]