Team:Aix-Marseille/Protocol:electrocompetent ecoli cells
From 2014.igem.org
(Difference between revisions)
(Created page with "{{Team:Aix-Marseille/header}} <html> <div class="container"> <div class="panel panel-info" id="panel-protocol"> <div class="panel-heading"> <div class="panel...") |
|||
Line 11: | Line 11: | ||
<h2>Preparation of competent cells</h2> | <h2>Preparation of competent cells</h2> | ||
<li> | <li> | ||
- | Streak E.coli cells | + | Streak E.coli cells on an LB plate with or without antibiotics. |
</li> | </li> | ||
<li> | <li> | ||
Line 29: | Line 29: | ||
</li> | </li> | ||
<li> | <li> | ||
- | Centrifuge cells in Sorval GSA rotor at 4°C for 10 mins at 5 000 | + | Centrifuge cells in Sorval GSA rotor at 4°C for 10 mins at 5 000 rpm. |
</li> | </li> | ||
Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room. | Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room. | ||
<li> | <li> | ||
- | Discard the supernatant and resuspend cells in 1 Vol ice-cold sterile 10% glycerol (gentle shaking on ice or in the coldroom is the best way to go). Spin the cells at 6000 | + | Discard the supernatant and resuspend cells in 1 Vol ice-cold sterile 10% glycerol (gentle shaking on ice or in the coldroom is the best way to go). Spin the cells at 6000 rpm at 4°C for 10 mins. |
</li> | </li> | ||
<li> | <li> | ||
Line 50: | Line 50: | ||
<h2>Transformation</h2> | <h2>Transformation</h2> | ||
<li> | <li> | ||
- | To use the frozen cells, thaw on ice the individual aliquots, add salt-free DNA to them (typically 1-5 µl), mix by pipetting, and transfer the mix to a pre-chilled electroporation cuvette. | + | To use the frozen cells, thaw on ice the individual aliquots, add salt-free DNA to them (typically 1-5 µl), mix by pipetting, and transfer the mix to a pre-chilled electroporation cuvette on ice. Avoid introducing air bubbles into the cell mix in the cuvette as this can cause arcing during electroporation. |
</li> | </li> | ||
<li> | <li> | ||
- | + | Activate electroporator by pressing the ON/STDBY key. For BioRad system, set on EC1. | |
</li> | </li> | ||
<li> | <li> | ||
- | + | Bring the ice bucket with cuvettes in it, plus SOC and P1000 pipette close to the electroporator. | |
</li> | </li> | ||
<li> | <li> | ||
- | + | Remove the electroporation cuvette containing the competent cells/DNA mix from the ice, wipe off any excess ice/moisture from the cuvette and place it into the electroporator cuvette holder (the cuvettes have a notch protruding from them and as such can only fit into the cuvette holder in one direction). | |
+ | </li> | ||
+ | <li> | ||
+ | Slide the cuvette holder all the way back until it is flush with the front of the unit. | ||
+ | </li> | ||
+ | <li> | ||
+ | Press the PULSE key. A “beep” will sound indicating that electroporation has occurred. | ||
+ | </li> | ||
+ | <li> | ||
+ | Immediately remove the cuvette and add 650 µL SOC (or LB if you are out of SOC). | ||
+ | </li> | ||
+ | <li> | ||
+ | Outgrow (recover) the transformed cells by gentle shaking at 37°C for ~1h. | ||
+ | </li> | ||
+ | <li> | ||
+ | Plate a serial dilution of the transformed cells on selective medium. | ||
</li> | </li> | ||
</ol> | </ol> |
Latest revision as of 19:40, 15 July 2014
Electrocompetent Escherichia coli cells
- Streak E.coli cells on an LB plate with or without antibiotics.
- Allow cells to grow at 37°C overnight.
- Place three or four colonies in 5 mL LB media (+ antibiotic selection if necessary), grow overnight at 37°C.
- Dilute the preculture to OD600 = 0,005 in 100 mL LB.
- Allow cell to grow at 37°C (250 rpm), until OD600= 0.4 (about 2-3 hours).
- Transfer cells to 2 centrifuge bottles (50 mL), and place cells on ice for 20 mins.
- Centrifuge cells in Sorval GSA rotor at 4°C for 10 mins at 5 000 rpm. Subsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room.
- Discard the supernatant and resuspend cells in 1 Vol ice-cold sterile 10% glycerol (gentle shaking on ice or in the coldroom is the best way to go). Spin the cells at 6000 rpm at 4°C for 10 mins.
- Resuspend the pellet in 0.5 Vol of ice-cold sterile 10% glycerol. Spin at 8000 rpm at 4°C for 15 mins (after washing, the cells become harder and harder to pellet down).
- Wash the cells with ~20mL (per liter of the original cell culture) of ice-cold sterile 10% glycerol and transfer the cell suspension to a pre-chilled small centriguge tube. Spin the cells at 10000 tr/min at 4°C for 15 mins.
- Resuspend the cells in 2-3 ml of ice-cold sterile 10% glycerol per liter of the original cell culture.
- Aliquot the cells into pre-chilled microfuge tubes (40 µl per transformation in a 0.1-0.2 cm gap electroporation cuvette). The cells are now ready to use. For long-term storage, store at -80°C the tubes with 50 µl aliquots.
- To use the frozen cells, thaw on ice the individual aliquots, add salt-free DNA to them (typically 1-5 µl), mix by pipetting, and transfer the mix to a pre-chilled electroporation cuvette on ice. Avoid introducing air bubbles into the cell mix in the cuvette as this can cause arcing during electroporation.
- Activate electroporator by pressing the ON/STDBY key. For BioRad system, set on EC1.
- Bring the ice bucket with cuvettes in it, plus SOC and P1000 pipette close to the electroporator.
- Remove the electroporation cuvette containing the competent cells/DNA mix from the ice, wipe off any excess ice/moisture from the cuvette and place it into the electroporator cuvette holder (the cuvettes have a notch protruding from them and as such can only fit into the cuvette holder in one direction).
- Slide the cuvette holder all the way back until it is flush with the front of the unit.
- Press the PULSE key. A “beep” will sound indicating that electroporation has occurred.
- Immediately remove the cuvette and add 650 µL SOC (or LB if you are out of SOC).
- Outgrow (recover) the transformed cells by gentle shaking at 37°C for ~1h.
- Plate a serial dilution of the transformed cells on selective medium.
Preparation of competent cells
Transformation