Difference between revisions of "Electroporation Transformation"
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'''Cell Preparation''' | '''Cell Preparation''' | ||
| − | 1. This procedure yields enough bacteria for two transformations | + | 1. This procedure yields enough bacteria for two transformations - scale up for more |
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2. Grow bacteria to be transformed in 40 ml of broth in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight) | 2. Grow bacteria to be transformed in 40 ml of broth in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight) | ||
| + | |||
3. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH2O | 3. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH2O | ||
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4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O | 4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O | ||
| + | |||
5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O | 5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O | ||
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6. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O | 6. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O | ||
| + | |||
7. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 80 ul sterile 10% glycerol (optional: 72 ul sterile dH2O and 8 ul sterile glycerol) | 7. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 80 ul sterile 10% glycerol (optional: 72 ul sterile dH2O and 8 ul sterile glycerol) | ||
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1. Set charging voltage to 1.3 - 1.5 kV | 1. Set charging voltage to 1.3 - 1.5 kV | ||
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2. Pipette 1 ul of plasmid (1 ng/ul) into the electroporation cuvette | 2. Pipette 1 ul of plasmid (1 ng/ul) into the electroporation cuvette | ||
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3. Add 40 ul of electrocompetent bacteria to the cuvette and flick gently | 3. Add 40 ul of electrocompetent bacteria to the cuvette and flick gently | ||
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4. Click the Pulse button | 4. Click the Pulse button | ||
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5. Transfer to sterile 1.5 ml tube and immediately add 960 ul sterile LB or SOC | 5. Transfer to sterile 1.5 ml tube and immediately add 960 ul sterile LB or SOC | ||
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6. Incubate at 37 C with shaking for 1 hour | 6. Incubate at 37 C with shaking for 1 hour | ||
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7. Spin in microfuge for 30 seconds | 7. Spin in microfuge for 30 seconds | ||
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8. Add 50 ul LB + antibiotic and spread onto agar plate with antibiotic | 8. Add 50 ul LB + antibiotic and spread onto agar plate with antibiotic | ||
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1. Fill the cuvettes two times with dH2O, dumping each time | 1. Fill the cuvettes two times with dH2O, dumping each time | ||
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2. Fill the cuvettes with 0.1 N NaOH and let sit for 5 minutes | 2. Fill the cuvettes with 0.1 N NaOH and let sit for 5 minutes | ||
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3. Fill the cuvettes two times with dH2O, dumping each time | 3. Fill the cuvettes two times with dH2O, dumping each time | ||
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4. Fill the cuvettes two times with 95% ethanol, dumping each time | 4. Fill the cuvettes two times with 95% ethanol, dumping each time | ||
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5. Turn upside down to dry | 5. Turn upside down to dry | ||
Revision as of 14:38, 10 March 2014
This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA.
Cell Preparation
1. This procedure yields enough bacteria for two transformations - scale up for more
2. Grow bacteria to be transformed in 40 ml of broth in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight)
3. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH2O
4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O
5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH2O
6. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH2O
7. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 80 ul sterile 10% glycerol (optional: 72 ul sterile dH2O and 8 ul sterile glycerol)
Electroporation
1. Set charging voltage to 1.3 - 1.5 kV
2. Pipette 1 ul of plasmid (1 ng/ul) into the electroporation cuvette
3. Add 40 ul of electrocompetent bacteria to the cuvette and flick gently
4. Click the Pulse button
5. Transfer to sterile 1.5 ml tube and immediately add 960 ul sterile LB or SOC
6. Incubate at 37 C with shaking for 1 hour
7. Spin in microfuge for 30 seconds
8. Add 50 ul LB + antibiotic and spread onto agar plate with antibiotic
Washing Electroporation Cuvettes
1. Fill the cuvettes two times with dH2O, dumping each time
2. Fill the cuvettes with 0.1 N NaOH and let sit for 5 minutes
3. Fill the cuvettes two times with dH2O, dumping each time
4. Fill the cuvettes two times with 95% ethanol, dumping each time
5. Turn upside down to dry
