Bacterial Transformation Principle

Question 1

Procedure
1 Thaw Competent Cells

Answer 1

Thaw 50–100 μL of competent E. coli cells on ice. Keep them on ice throughout the process to prevent cell damage.

Question 2

Add Plasmid DNA:

Answer 2

• Add 1–5 μL of plasmid DNA (10–100 ng) to the competent cells.
• Gently mix the contents by flicking the tube or lightly tapping it. Do not vortex. Incubate on ice for 15–30 minutes.

Question 3

Heat Shock:

Answer 3

• Place the tube of cells in a 42°C water bath for 30 seconds. This creates a heat shock
  that helps the plasmid enter the cells.
• After 30 seconds, immediately place the tube back on ice for 2–5 minutes to let the cells recover.

Question 4

Add LB Broth:

Answer 4

• Add 250–500 μL of LB or to the tube to help the cells recover from the heat shock.
• Incubate the tube at 37°C for 30–60 minutes. This allows the bacteria to recover and start expressing the antibiotic resistance gene.

Question 5

Plate the Cells:

Answer 5

• After incubation, plate 50–100 μL of the transformation mixture onto an LB agar plate containing the appropriate antibiotic (e.g., ampicillin).
• Spread the liquid evenly across the plate using a sterile loop or spreader.
• (Optional) If you want more colonies, you can centrifuge the remaining mixture, remove some supernatant, resuspend in a smaller volume, and plate more cells.

Question 6

Incubate:

Answer 6

• Invert the plates and incubate them at 37°C overnight (12–16 hours).

Question 7

Check for Colonies:

Answer 7

• After incubation, check the plates for colony growth. Only transformed bacteria will
  grow on the antibiotic-containing plates.

Question 8

• _ and _ should always be kept on ice to preserve their integrity.
• Handle the competent cells _ to avoid damaging them.
• _ will ensure that only transformed bacteria grow on the plates.

Answer 8

Competent cells and plasmid DNA
- gently
- Antibiotic selection