Erickson: Electroporation

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Prepare 5 mL of bacterial strain that you wish to transform in BHI.
Prepare 5 mL of bacterial strain that you wish to transform in BHI.
 +
Grow overnight .
Grow overnight .
 +
Chill sterile microfuge tubes on ice and turn on the refridgerated centrifuge.
Chill sterile microfuge tubes on ice and turn on the refridgerated centrifuge.
 +
Add 1 ½ mL of the liquid culture to each microfuge tube until the culture is gone.  Keep on ice.
Add 1 ½ mL of the liquid culture to each microfuge tube until the culture is gone.  Keep on ice.
 +
Centrifuge for 2-3 minutes at 13,200 rpm.
Centrifuge for 2-3 minutes at 13,200 rpm.
 +
Remove supernatant completely.
Remove supernatant completely.
 +
Add 1 mL COLD glycerol to each tube.  Resuspend pellet entirely.
Add 1 mL COLD glycerol to each tube.  Resuspend pellet entirely.
 +
Centrifuge again.   
Centrifuge again.   
 +
Remove supernatant and resuspend in 0.5 mL glycerol
Remove supernatant and resuspend in 0.5 mL glycerol
 +
Remove supernatant and resuspend in 0.25 mL glycerol.
Remove supernatant and resuspend in 0.25 mL glycerol.
 +
Remove supernatant and resuspend in 0.25 mL glycerol.
Remove supernatant and resuspend in 0.25 mL glycerol.
-
Remove supernatant and resuspend in 100 µL glycerol, combining all tubes into one (resuspend one pellet and use that solution to resuspend the next).
+
 
 +
Remove supernatant and resuspend in 100 µL glycerol, combining all tubes into one (resuspend one pellet and use that solution  
 +
to resuspend the next).
 +
 
Centrifuge.
Centrifuge.
 +
Resuspend pellet in 25µL glycerol.  Keep on ice until you add DNA for the transformation.
Resuspend pellet in 25µL glycerol.  Keep on ice until you add DNA for the transformation.
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Thaw electrocompetent cells on ice (if necessary).
Thaw electrocompetent cells on ice (if necessary).
 +
Chill electroporation cuvettes on ice.   
Chill electroporation cuvettes on ice.   
 +
Add 5µL purified plasmid to electrocompetent cells.  Swirl with pipette tip to mix.
Add 5µL purified plasmid to electrocompetent cells.  Swirl with pipette tip to mix.
 +
Transfer cells with plasmid to cuvette.
Transfer cells with plasmid to cuvette.
 +
Pulse once on setting Ec1.
Pulse once on setting Ec1.
 +
Immediately add 500µL SOC broth.  Transfer cells to a new tube.  Incubate with shaking for 1-2 hours.
Immediately add 500µL SOC broth.  Transfer cells to a new tube.  Incubate with shaking for 1-2 hours.
 +
Wash cuvettes.  Soak them in ethanol overnight, rinse them with water, and let them dry under the bio-hood.
Wash cuvettes.  Soak them in ethanol overnight, rinse them with water, and let them dry under the bio-hood.
 +
Plate the transformed bacteria on selective media, 100 µL on each plate.  Incubate.
Plate the transformed bacteria on selective media, 100 µL on each plate.  Incubate.

Revision as of 14:35, 23 June 2008

Transformation by Electroporation

Competent cells

Prepare 5 mL of bacterial strain that you wish to transform in BHI.

Grow overnight .

Chill sterile microfuge tubes on ice and turn on the refridgerated centrifuge.

Add 1 ½ mL of the liquid culture to each microfuge tube until the culture is gone. Keep on ice.

Centrifuge for 2-3 minutes at 13,200 rpm.

Remove supernatant completely.

Add 1 mL COLD glycerol to each tube. Resuspend pellet entirely.

Centrifuge again.

Remove supernatant and resuspend in 0.5 mL glycerol

Remove supernatant and resuspend in 0.25 mL glycerol.

Remove supernatant and resuspend in 0.25 mL glycerol.

Remove supernatant and resuspend in 100 µL glycerol, combining all tubes into one (resuspend one pellet and use that solution to resuspend the next).

Centrifuge.

Resuspend pellet in 25µL glycerol. Keep on ice until you add DNA for the transformation.


Transformation

Thaw electrocompetent cells on ice (if necessary).

Chill electroporation cuvettes on ice.

Add 5µL purified plasmid to electrocompetent cells. Swirl with pipette tip to mix.

Transfer cells with plasmid to cuvette.

Pulse once on setting Ec1.

Immediately add 500µL SOC broth. Transfer cells to a new tube. Incubate with shaking for 1-2 hours.

Wash cuvettes. Soak them in ethanol overnight, rinse them with water, and let them dry under the bio-hood.

Plate the transformed bacteria on selective media, 100 µL on each plate. Incubate.

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