Talk:Team:IvyTech-South Bend/Notebook/4 August 2010

From 2010.igem.org

Plate 3 well 22C BBa_K131010- A/K resistance

1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.

2. Add 10uL of diH2O (deionized water)

3. Pipette 1 or 2uL of the resuspended DNA transform into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.

4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.

5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

   * To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance. 

This protocol is for the typical electro-transformation of E. coli done in the Richard Lab.

The consensus electroporation protocol should be consulted if deviating from the procedure outlined here. Procedure

1. Chill the # electroporation cuvettes by floating them in an ice bath.

2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.

3. Prepare # micro-centrifuge tubes containing 900μl SOB media.

4. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes).

5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.

6. Place cells back on ice to ensure they remain cold.

7. Pippette 100μL of cell-DNA mixture to cuvette.

8. Wipe off excess moisture from outside of cuvette.

9. Place cuvette in chamber of electroporator.

10. Pulse the cells by pressing button on electroporator twice.

11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.

12. Let cells recover at room temperature for 30-60 minutes.

13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C. --Rchamberlin 18:00, 6 August 2010 (UTC)