Difference between revisions of "Electroporation Transformation"
(Created page with 'This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA. '''Cell Preparation''' 1. This procedure yields enough bacteria for two transformati…') |
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| − | This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA | + | '''This procedure uses electroporation to achieve transformation of bacteria with plasmid DNA''' |
| + | |||
'''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 |
| − | 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 | + | 2. Grow bacteria to be transformed in 40 ml of broth (eg. LB Broth) in a 50 ml tuble, allowing them to grow to an OD590 of 0.5 to 1.0 (optional: just grow overnight) |
| − | 4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile | + | |
| − | 5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in | + | 3. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 40 ml sterile dH<sub>2</sub>O |
| − | 6. | + | |
| − | + | 4. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 20 ml sterile dH<sub>2</sub>O | |
| + | |||
| + | 5. Pellet the culture by centrifuging at 4000xg for 5 minutes; resuspend in 800 ul sterile dH<sub>2</sub>O | ||
| + | |||
| + | 6. Transfer to a sterile 1.5 mL tube and pellet the culture by centrifuging in a microfuge for 1 minute; resuspend in 80 ul sterile 10% glycerol | ||
| + | |||
'''Electroporation''' | '''Electroporation''' | ||
| − | 1. | + | 1. Use a clean electroporation cuvette with chamber gap of 1 mm and set charging voltage to 1.3 - 1.5 kV |
| + | |||
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 | ||
| + | |||
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 | ||
| − | 4. Click the Pulse button | + | |
| + | 4. Click the Pulse button. If there is too much salt, the cuvette can arch and make a pop sound. [https://vimeo.com/user37250198/review/172478413/c3537bdf0e see example] | ||
| + | |||
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 | ||
| + | |||
6. Incubate at 37 C with shaking for 1 hour | 6. Incubate at 37 C with shaking for 1 hour | ||
| − | 7. Spin in microfuge for | + | |
| − | 8. Add 50 ul LB + antibiotic and spread onto agar plate with antibiotic | + | 7. Spin in microfuge for 60 seconds, pour off all supernatant |
| + | |||
| + | 8. Add 50 ul LB + antibiotic, resuspend pellet, and spread onto agar plate with antibiotic | ||
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'''Washing Electroporation Cuvettes''' | '''Washing Electroporation Cuvettes''' | ||
| − | 1. Fill the cuvettes two times with | + | 1. Fill the cuvettes two times with dH<sub>2</sub>O, dumping each time |
| + | |||
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 | ||
| − | 3. Fill the cuvettes two times with | + | |
| + | 3. Fill the cuvettes two times with dH<sub>2</sub>O, dumping each time | ||
| + | |||
4. Fill the cuvettes two times with 95% ethanol, dumping each time | 4. Fill the cuvettes two times with 95% ethanol, dumping each time | ||
| + | |||
5. Turn upside down to dry | 5. Turn upside down to dry | ||
Latest revision as of 13:46, 21 January 2018
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 (eg. LB 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 800 ul sterile dH2O
6. Transfer to a sterile 1.5 mL tube and pellet the culture by centrifuging in a microfuge for 1 minute; resuspend in 80 ul sterile 10% glycerol
Electroporation
1. Use a clean electroporation cuvette with chamber gap of 1 mm and 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. If there is too much salt, the cuvette can arch and make a pop sound. see example
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 60 seconds, pour off all supernatant
8. Add 50 ul LB + antibiotic, resuspend pellet, 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
