Richard Lab:Electroporation of E. coli
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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.
- Chill electroporation cuvettes and DNA samples on ice.
- Remove # vials containing 100μl electro-competent cells from the -80°C freezer. Thaw the vials on ice. Be sure to include 2 vials for a negative and an unligated vector control.
- Prepare # micro-centrifuge tubes containing 900μl SOB media.
- Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes)
- 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.
- Place cells back on ice to ensure they remain cold.
- Pippette 100μL of cell-DNA mixture to cuvette.
- Wipe off excess moisture from outside of cuvette.
- Place cuvette in chamber of electroporator.
- Pulse the cells by pressing button on electroporator twice.
- Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.
- Let cells recover at room temperature for 30-60 minutes.
- Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.
- Leave remaining SOB-cell mixture on the bench-top overnight.
- If you don't have any transformants in the morning then plate the rest of the transformation.
- Our "1X TBE" is technically 0.5X TBE (5.3g/L Tris Base, 2.75g/L Boric Acid, and 2.9g/L EDTA) but for our purposes we call it 1X TBE.
- Correspondingly, the 10X TBE is officially 5X.
- Using TBE allows us to have such a high (150V) voltage. If you use TAE you need to use a significantly lower voltage and your run time will be longer. Despite that, TAE is advantagous in some cases, but Mike feels that TBE is better suited for his applications. This is discussed in detail in the consensus protocol.
- Use the following table to determine the amount of agarose you want to use.
|Agarose Concentration (g/100mL)||Optimal DNA Resolution (kb)|
|0.5||1 - 30|
|0.7||0.8 - 12|
|1.0||0.5 - 10|
|1.2||0.4 - 7|
|1.5||0.2 - 3|
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