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From 2010.igem.org

10/26/10

Yesterday was a huge discovery. E.coli does not produce AHL so we were testing the wrong bacteria. Pseudomonas produce AHL so we belive that a contamination happened to produce the blue on the electroporated E.coli. We have a working part so we electroporated E.coli, A.Tumefaciena and mycobateria with the dna from TCN backbone

cut @ E,P LacZ which is cut @ E,S and T9002 cut @E,X Ligation and electroporation was a success.

We will now purify/extract DNA then relegate to remove (TNC) backbone and replace with Chloramphenicol backbone and suspend into LB/ Chloramphenicol broth for keeping our part and to ship to IGem HQ . We are sending a ligation mixture of TCN, Amp, Chloramphenicol backbones. We will be shipping 25ml of this sample this afternoon. Dylan will purify DNA using PCRKleen Spin Columns following protocol from Kit

2.1 Instructions for Use

1. Resuspend the resin in the column by vortexing -5 seconds.

2. Remove the cap, snap off the tip and place the column in a 2.0 ml , wash tube

3. Pre-spin the column for 1 minute in a microcentrifunge at 735 x g. (If used at higher g forces, results may vary)

4. Place the column in a clean 1.5 ml collection tube

5. Apply the sample (25-100 ul) to the top center of the column bed, being careful not to disturb the resin

6. Spin the column for 2 minutes at 735 g.

7. Save the purified sample which is in the bottom of the 1.5 ml collection tube.

8. Properly dispose of the used column which contains unincorporated dNTP’s, primers, and short primer-dimers.

Quantum Prep PCR Kleen spin columns fit 1.5 ml and 2.0 ml microcentrifuge rubes for sample collection during centrifugation. Use the 2.0 ml capless wash tubes for initial column buffer removal, and the 1.5 ml collection tubes for isolation of purified samples. The tubes which come \ :si -’~ with the columns are completely autoclavable, and should be autoclaved if sterile tubes are desired.

PCR Kleen spin columns are designed to be used in variable speed bench top microcentrifuges capable of generating a force of 735 x g. The ~avitational force created at a particular rpm is a function of the radius of the microcentrifuge rotor. Consult the microcentrifuge instruction manual for conversion information from rpm to g-force. Alternatively, to calculate the speed (rpm) required to reach a D’avitational force of 735 x g, use the following equation.

RCF (g) = (1.12 X 10-5)(rpm)2r

where r is the radius in centimeters measured from the center of the rotor to the middle of the PCR Kleen column, and rpm is the speed of the rotor in revolutions per minute.

Note: Do not use the pulse button to centrifuge the columns--this overo rides the variable speed setting and can cause column failure.

If a variable speed microcentrifuge is not available, PCR Kleen spin columns. can be spun in a clinical centrifuge set at 735 x g. Place the " column in a collection tube, and set the column and collection tube in a larger tube appropriate for the size of the rotor. Centrifuge at 735 x g for the times specified in the Instructions for Use section.

SAMPLE Sent

overnight Fedx 9:00 am delivery @3:00 pm

--for JHullRchamberlin 15:20, 27 October 2010 (UTC)