IG Recombineering Electrocompetent Competent Cells

Calculation of Transformation Efficiency:

Transformation Efficiency (TE) is defined as the number of colony forming units (cfu) produced by transforming 1µg of plasmid into a given volume of competent cells.

TE = Colonies/µg/Dilution

Transform 1 µl of (10 pg/µl) pUC19 control plasmid into 50 µl of cells, add 950 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 100 colonies, the TE is calculated as follows:

Colonies = 100
µg of DNA = 0.00001
Dilution = 50/1000 x 10/1000 = 0.0005
TE = 100/.00001/.0005 = 2.0×10¹⁰

Transformation Protocol:

Use this procedure to transform IG® Recombineering Electrocompetent Cells. Do not use these cells for chemical transformation.

1. DNA/Cell Mixture Preparation

1.1. Remove competent cells from the -80 °C freezer and thaw completely on wet ice (10–15 minutes). Place sterile electroporation cuvettes and microcentrifuge tubes on ice. Bring IG Recovery Medium to room temperature.

1.2. Aliquot 1–5 µl DNA (1 pg–100 ng) into chilled microcentrifuge tubes on ice. If using the pUC19 control, add 1 µl pUC19 DNA (10 pg/µl) to a chilled microcentrifuge tube.

1.3. Once the cells are thawed, gently add 25 µl of competent cells to each DNA tube while keeping the mixture on ice. Mix gently by tapping the tube 4–5 times.

Do not pipette up and down or vortex, as this may damage the cells and reduce transformation efficiency.

2. Electroporation

2.1. Pipette 26 µl of the cell/DNA mixture into a chilled electroporation cuvette, avoiding bubbles. Quickly flick the cuvette downward to ensure the mixture settles evenly across the bottom of the cuvette, then proceed with electroporation.

Standard electroporation settings for E. coli:
Voltage: 1.8 kV, Resistance: 200 Ω, Capacitance: 25 µF, Cuvette gap: 0.1 cm.

3. Cell Recovery

3.1. Immediately add 974 µl of room-temperature IG Recovery Medium (or another suitable medium) to the cuvette. Gently pipette up and down three times to resuspend the cells.

3.2. Transfer the entire mixture to a culture tube (17 mm × 100 mm) and incubate in a 30 °C shaking incubator at 210 rpm for 1 hour. (Do NOT grow this cell and its transformants at > 32°C)

4. Cell Plating

4.1. Prepare pre-warmed selection plates during the recovery period. Plates should be equilibrated to 30 °C and free of condensation to prevent contamination and mixed colonies.

4.2. Dilute the recovered cells 10–100X, if necessary, to obtain well-isolated colonies. Plate 20–200 µl of cells onto pre-warmed LB agar plates containing the appropriate antibiotic(s). For the pUC19 control, plate 50 µl of a 100X diluted transformants onto an LB plate containing 100 µg/ml ampicillin. Spread the cells evenly using a sterile spreader or autoclaved ColiRoller™ plating beads.

4.3. Incubate the plates overnight (12–16 hours) at 30 °C.

References on How Recombineering Works:

In addition to selection, successful transformation of plasmids or vectors into IG® Recombineering Chemically Competent Cells can be verified by plasmid/vector identification and/or PCR. The references below describe how to use a transformed and confirmed clone carrying your target plasmid or vector in IG® Recombineering Electrocompetent Cells.

• Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005 Feb 24;33(4):e36. doi: 10.1093/nar/gni035. PMID: 15731329; PMCID: PMC549575.
• Sawitzke JA, Thomason LC, Bubunenko M, Li X, Costantino N, Court DL. Recombineering: highly efficient in vivo genetic engineering using single-strand oligos. Methods Enzymol. 2013;533:157-77. doi: 10.1016/B978-0-12-420067-8.00010-6. PMID: 24182922; PMCID: PMC7518103.