Transformation of DNA

Preparation of competent E. coli cells

Cells that can take up DNA (from various sources) are termed “competent”. Several techniques exist to prepare competent cells and one such technique for preparing competent E. coli is given below.

Note: Cells prepared using this protocol are not suitable for electroporation.

Materials required:

• E. coli cells in glycerol stock vial
• LB medium
• LB-agar plates
• Appropriate selective antibiotics
• TFB1 buffer (see table, Buffer TFB1)
• TFB2 buffer (see table, Buffer TFB2)

Buffer TFB1

Buffer TFB2

1. Remove a trace of E. coli cells from the glycerol stock vial with a sterile toothpick or inoculating loop, and streak it out on LB-agar plates containing an appropriate concentration of the relevant selective antibiotic(s) (see Antibiotics). If the host strain has already been cultured and stored at 2–8°C (cultures can be stored at 2–8°C for up to 3 months without any significant loss of viability), streak out bacteria from those stocks.
2. Incubate at 37°C overnight.
3. Pick a single colony and inoculate 10 mL LB medium containing relevant antibiotic(s). Grow overnight at 37°C
4. Add 1 mL overnight culture to 100 mL prewarmed LB medium containing the relevant antibiotic(s) in a 500 mL flask, and shake at 37°C until an OD600 of 0.5 is reached (approximately 90–120 min).
5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube
6. Collect the cells by centrifugation at low speed (5 min, 4000 x g, 4°C).
7. Discard the supernatant carefully. Always keep the cells on ice.
8. Resuspend the cells gently in cold (4°C) TFB1 buffer (30 mL for a 100 mL culture) and keep the suspension on ice for an additional 90 min.
9. Collect the cells by centrifugation (5 min, 4000 x g, 4°C).
10. Discard the supernatant carefully. Always keep the cells on ice.
11. Resuspend the cells carefully in 4 mL ice-cold TFB2 buffer.
12. Prepare aliquots of 100–200 µL in sterile microcentrifuge tubes and freeze in liquid nitrogen or a dry-ice–ethanol mix. Store the competent cells at –70°C.

Transformation involves introducing plasmid DNA into a bacterial host cell. Several methods exist for transforming bacterial cells; one common method is given below.

• Competent E. coli cells (see Preparation of competent E. coli)
• SOC medium (see table SOC medium)
• LB-agar plates (see Solid media)

SOC medium

1. Transfer an aliquot of the DNA to be transformed (10 µL or less) into a cold, sterile 1.5 mL microcentrifuge tube, and keep it on ice.
2. Thaw an aliquot of frozen competent E. coli cells on ice.
3. Gently resuspend the cells and transfer 100 µL of the cell suspension into the microcentrifuge tube with the plasmid DNA, mix carefully, and keep on ice for 20 min.
4. Transfer the tube to a 42°C water bath or heating block for 90 s
5. Add 500 µL SOC medium to the cells and incubate for 60–90 min at 37°C.
   Tip: Shaking increases transformation efficiency.
6.  Plate out 50, 100, and 200 µL aliquots on LB-agar plates containing the relevant antibiotic(s). Incubate the plates at 37°C overnight until colonies develop.