Transformation¶
After assembly, the circular plasmid has been generated, but it is mixed with other DNAs—some incomplete or mutant. By passing the material through cells, we isolate a single one of these sequences and amplify it a billion-fold.
Transformation is the process in which a bacterium takes up DNA from the environment. While some bacteria (like B. subtilis) do this naturally, E. coli requires preparation to become “competent.”
There are two common methods:
🔌 Electroporation¶
Electroporation involves preparing cells in salt-free, ice-cold water or 10% glycerol and shocking them with a brief electric pulse in a special cuvette. This creates pores in the membrane, allowing DNA to enter. It is:
- ~100× more efficient than heat shock
- More expensive (cuvettes are single-use)
- Sensitive to salt contamination
- Ideal for library transformations needing high colony counts
🌡️ Heat Shock (used in pP6)¶
We use the TSS method for heat shock, where cells are suspended in a PEG-salt mix. You add DNA and a small amount of KCM buffer, chill, then heat shock at 42°C for 90 seconds.
📄 Full TSS protocol
TSS cells can be frozen in aliquots and remain competent for years. TSS cells are easier to work with during transformation and are well-suited for routine cloning workflows.
In dilute conditions, as in pP6, most cells take up just one plasmid, resulting in unique colonies.
Rescue Step?¶
Some antibiotics (e.g., kanamycin, chloramphenicol) block translation immediately. To survive, cells must first express the resistance gene—this requires a rescue step (1 hour outgrowth in rich media before plating).
Ampicillin, used in pP6, is different: it kills only actively growing cells. Cells recover naturally after transformation, so no rescue is required.
Amp vs. Carbenicillin¶
Both are β-lactam antibiotics. The bla gene on pP6 confers resistance to both.
- Ampicillin breaks down when heated (e.g., during plate pouring)
- Carbenicillin is more stable, so we use Carb in practice
Cells without plasmid die at 5 µg/mL; with plasmid, they survive over 1000 µg/mL—a wide window for selection.
Transformation Efficiency¶
Transformation efficiency measures how effectively DNA is taken up and expressed by cells. It's typically calculated as:
CFU/µg DNA = (Colonies on plate) ÷ (amount of plasmid DNA in µg)
Our heat shock competent cells typically yield around 10⁶ CFU/µg DNA. With electroporation, efficiencies as high as 10¹¹ CFU/µg DNA can be achieved.
This varies based on:
- Method (electroporation ≫ heat shock)
- Competency of the cells
- DNA purity and volume
- Handling technique
Don’t worry about calculating it for pP6, but understand that low DNA input typically gives 1 DNA per cell, which is ideal when selecting single clones.
Handling Competent Cells¶
Competent cells are fragile. To maintain their viability:
- Keep them cold at all times (on ice or in a cold block).
- Do not pipette vigorously. Stir gently with the tip, then pipette up and down once or twice carefully.
- Avoid bubbles—they indicate lysis.
- Aliquot immediately after prep—cells degrade with freeze-thaw cycles.
- Do not re-freeze thawed competent cells—even one additional freeze-thaw cycle can dramatically reduce their transformation efficiency.
Note: You will not make your own competent cells. We prepare large batches once a year using the TSS method and store them at -80°C. Your instructor or supervisor will provide a frozen aliquot when needed.
Plating Techniques¶
Once transformation is complete, you’ll plate your cells using one of two methods:
1. Glass beads:
- Pour the transformation mix onto the plate.
- Add ~5–10 sterile glass beads.
- Gently shake the plate in a circular motion (avoid sloshing).
- Pour off the beads into the discard jar.
2. Metal spreader:
- Use a sterile spreader. These can be disposable plastic, reusable metal or glass wands, or made by bending a glass pipette. We use bent coat hangers shaped into a triangle—they cool quickly, are unbreakable, and can be reused indefinitely.
- Flame sterilize, cool briefly.
- Spread the liquid evenly by rotating the plate under the spreader.
- Don't gouge the agar.
Invert the plate (lid on the bottom) and place it in the incubator (not a shaker)
Labeling and Inventory¶
Be consistent and thorough:
- Write label, date, and your name on the plate bottom (not the lid since those can fall off).
- Match plate name to your sample ID and lab sheet entry (e.g.,
pP6-79). - Return any used DNA tubes to their storage rack and note usage if needed.