Transform competent E. coli cells: Difference between revisions
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Transform competent E. coli cells | Transform competent E. coli cells | ||
1.Prepare for transformation by ensuring the following: | 1. Prepare for transformation by ensuring the following: | ||
* Water bath is set to 42°C | * Water bath is set to 42°C | ||
* Vial of SOC medium is warmed to room temperature | * Vial of SOC medium is warmed to room temperature | ||
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* Tubes of competent cells will be shaken horizontally which means cells will come into contact with the cap of the tube. To maintain good sterile technique and prevent contamination, do not touch the inside of the cap as you perform the transformation. | * Tubes of competent cells will be shaken horizontally which means cells will come into contact with the cap of the tube. To maintain good sterile technique and prevent contamination, do not touch the inside of the cap as you perform the transformation. | ||
2.Add 2 µL of the TOPO® Cloning reaction (or 1 µL of plasmid) into a vial of One Shot® Chemically Competent E. coli and mix gently. Do not mix by pipetting up and down–competent cells are fragile! | 2. Add 2 µL of the TOPO® Cloning reaction (or 1 µL of plasmid) into a vial of One Shot® Chemically Competent E. coli and mix gently. Do not mix by pipetting up and down–competent cells are fragile! | ||
3.Incubate on ice for 5–30 minutes. | 3. Incubate on ice for 5–30 minutes. | ||
4.Heat-shock the cells for 30-45 seconds at 42°C without shaking. | 4. Heat-shock the cells for 30-45 seconds at 42°C without shaking. | ||
5.Immediately transfer the tubes back to the ice. | 5. Immediately transfer the tubes back to the ice. | ||
6.Add 250 µL of room temperature S.O.C. medium. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour. | 6. Add 250 µL of room temperature S.O.C. medium. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour. | ||
7.Remove tube from the shaker and transfer 10 µL of incubated cells plus 20 µL of SOC medium to the surface of an LB/x-gal/kan selective plate and spread evenly across the surface with a sterile tool. | 7. Remove tube from the shaker and transfer 10 µL of incubated cells plus 20 µL of SOC medium to the surface of an LB/x-gal/kan selective plate and spread evenly across the surface with a sterile tool. | ||
8.Add 50 µL of incubated cells to a second LB/x-gal/kan plate and spread evenly across the surface with a sterile tool. | 8. Add 50 µL of incubated cells to a second LB/x-gal/kan plate and spread evenly across the surface with a sterile tool. | ||
9.To the non-selective LB plate, transfer 10 µL of incubated cells plus 20 µL of SOC medium and spread evenly across the surface with a sterile tool. | 9. To the non-selective LB plate, transfer 10 µL of incubated cells plus 20 µL of SOC medium and spread evenly across the surface with a sterile tool. | ||
10.If you want, plate the remainder of your incubated cells on a third LB/x-gal/kan plate and spread evenly across the surface with a sterile tool. | 10. If you want, plate the remainder of your incubated cells on a third LB/x-gal/kan plate and spread evenly across the surface with a sterile tool. | ||
11.Label the plates and place in the 37°C incubator overnight. | 11. Label the plates and place in the 37°C incubator overnight. | ||
12.Keep any unused cell culture in the 4°C fridge or coldroom (not freezer) | 12. Keep any unused cell culture in the 4°C fridge or coldroom (not freezer) | ||
13.The next day you will check your transformants. Patch at least 10 white (or light blue if necessary) colonies onto a fresh plate by selecting the colony with a sterile toothpick or inoculation loop and transferring it to a fresh LB/kan plate. Place the original plate at 4°C and the patched plate into the 37°C incubator overnight. | 13. The next day you will check your transformants. Patch at least 10 white (or light blue if necessary) colonies onto a fresh plate by selecting the colony with a sterile toothpick or inoculation loop and transferring it to a fresh LB/kan plate. Place the original plate at 4°C and the patched plate into the 37°C incubator overnight. | ||
Latest revision as of 12:02, 29 March 2023
Transform competent E. coli cells
1. Prepare for transformation by ensuring the following:
- Water bath is set to 42°C
- Vial of SOC medium is warmed to room temperature
- X-gal/Kan plates are warmed to 37°C
- One Shot® Chemically Competent E. coli are thawed on ice
- Tubes of competent cells will be shaken horizontally which means cells will come into contact with the cap of the tube. To maintain good sterile technique and prevent contamination, do not touch the inside of the cap as you perform the transformation.
2. Add 2 µL of the TOPO® Cloning reaction (or 1 µL of plasmid) into a vial of One Shot® Chemically Competent E. coli and mix gently. Do not mix by pipetting up and down–competent cells are fragile!
3. Incubate on ice for 5–30 minutes.
4. Heat-shock the cells for 30-45 seconds at 42°C without shaking.
5. Immediately transfer the tubes back to the ice.
6. Add 250 µL of room temperature S.O.C. medium. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour.
7. Remove tube from the shaker and transfer 10 µL of incubated cells plus 20 µL of SOC medium to the surface of an LB/x-gal/kan selective plate and spread evenly across the surface with a sterile tool.
8. Add 50 µL of incubated cells to a second LB/x-gal/kan plate and spread evenly across the surface with a sterile tool.
9. To the non-selective LB plate, transfer 10 µL of incubated cells plus 20 µL of SOC medium and spread evenly across the surface with a sterile tool.
10. If you want, plate the remainder of your incubated cells on a third LB/x-gal/kan plate and spread evenly across the surface with a sterile tool.
11. Label the plates and place in the 37°C incubator overnight.
12. Keep any unused cell culture in the 4°C fridge or coldroom (not freezer)
13. The next day you will check your transformants. Patch at least 10 white (or light blue if necessary) colonies onto a fresh plate by selecting the colony with a sterile toothpick or inoculation loop and transferring it to a fresh LB/kan plate. Place the original plate at 4°C and the patched plate into the 37°C incubator overnight.