Introducing foreign DNA into E. coli bacteria
Bacterial transformation is when cells take up foreign DNA from the environment. Cells may express the genetic information they received from this foreign DNA. (ThermoFisher)
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TIPS BEFORE STARTING:
- Depending on the situation, particularly when you get to the analysis phase and you request a different type of bacterial chassis, you will use a variety of competent cells. Be sure you know what cells you are using and if there is a specific protocol for the type of cell. (The competent bacteria described in this protocol are Zippy Competent JM109, ZymoResearch)
- Most plasmids carry a marker gene for a specific antibiotic resistance. By supplementing the growth medium with the antibiotic of choice, only cells containing the plasmid of interest will propagate. You must know the antibiotic resistance associated with your plasmid backbone!! (See Appendix I)
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All competent cells are stored in the -80 freezer. Ask the instructor for help when you are ready to get the competent cells. You must have ICE, first!
| Materials |
Storage Temperature |
| Zippy Competent Bacteria Cells |
-80°C |
| Ligation Mixture |
fresh / -20°C |
| LB Agar plate w/Antibiotic |
Room Temp / 37°C |
| SOC |
4°C / Room Temp |
| Sterile Glass Beads |
Room Temp |
Procedure: Read every step before you begin!
1) Make sure you have a warm plate. Pre-warm by placing it in the 37°C incubator or by taking it out at room temperature before starting the procedure. See color coding (Appendix I) to ensure you have appropriate LB+antibiotic plate type!
2) Get ice, then request cells / Let the cells thaw for 1-2 minutes on ice. – The 50 \(\mu L\) of cells can be split into 2 tubes of 25 \(\mu L\) each for two different transformations if used immediately to transform different plasmids. (If you don’t have two plasmids to transform, consider sharing with another team.)
3) Before the cells are completely thawed add 1-5 \(\mu L\) of ligation (plasmid) mixture directly to the cells (do not add to the side of the tube). For best results add the plasmid ligation to the cells while they are still slushy.
4) Incubate on ice for 5 minutes.
5) Add 200 \(\mu L\) of SOC media with no antibiotic to the cell-ligation mix.
(SOC amount = ~4X volume of cell + plasmid mix; thus only 120ul needed for 25ul transformation)
6). Incubate with gentle shaking (200-300 RPM) for 1-2 hrs at 37°C. (1 hr will work well but you get a higher transformation efficiency with 2 hr out-growths). Skip this step if using Ampicillin resistance and proceed directly to step 7.
7) Spread transformed cells using glass beads onto a LABELED warmed (22-37°C) plate containing antibiotic. Allow plates to dry (37°C) before flipping plates upside down. (You do not want media/bacteria dripping onto the lid. You can flip plates early the next day, if needed.)
8) Incubate overnight (18-20 hrs) at 37°C. The next day, remove plates from the incubator, seal the lids with parafilm and store at 4°C.
The next day: All colonies should be about the same size (diameter). It is not uncommon with 3A assembly to see only 1 to 10 colonies.
