IG Recombineering Chemically 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

High-Efficiency Transformation Protocol:

Use this procedure to transform IG® Recombineering Chemically Competent Cells.

These cells are designed for chemical transformation only. Do NOT use for electroporation. For electroporation transformation, use IG® Recombineering Electrocompetent Cells.

1. DNA/Cell Mixture Preparation

1.1. Remove competent cells from the –80 °C freezer and thaw completely on wet ice for 10–15 minutes. Bring IG Recovery Medium to room temperature.

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

1.3. Once thawed, gently add 50 µl of competent cells to each DNA tube while keeping on ice. Mix gently by tapping the tube 4–5 times.
If using the 50 µl package size competent cells, add DNA directly to the tube and mix gently by tapping.

Do not pipette up and down or vortex. Harsh mixing can damage cells and reduce transformation efficiency.

2. Chemical Transformation

2.1. Incubate the DNA/cell mixture on ice for 30 minutes.

2.2. Heat shock the cells by placing the culture tubes in a 42 °C water bath for 45 seconds.

2.3. Immediately return the tubes back to ice for 2 minutes.

3. Cell Recovery

3.1. Aliquot 950 µl of room-temperature Recovery Medium (or other suitable medium) into each culture tube (17 mm × 100 mm), then transfer the heat-shocked cell/DNA mixture into the corresponding tube.

3.2. Incubate the tubes in a shaking incubator at 30 °C by shaking at 210 rpm for 1 hour.

Do not grow this cell and its transformants at > 32°C.

4. Cell Plating

4.1. Plate 50–200 µl of each transformation onto pre-warmed LB agar plates containing the appropriate antibiotic(s). Spread evenly using a sterile spreader or autoclaved ColiRoller™ plating beads.

For the pUC19 control, plate 50 μl on an LB plate containing 100 μg/ml ampicillin.

Note: 

• Before cell plating, the plates should be prewarmed to growth temperature (37 °C), and be free of condensation to prevent contamination and mixed colonies.
• We recommend plating two different volumes to ensure well-isolated colonies on at least one plate.

4.2. Incubate the plates overnight (12-16 hr) at 37 °C.

Five Minute Transformation Protocol:

The following procedure results in only ~10% of the transformation efficiency as the protocol listed above.

1. Remove competent cells from the -80 °C freezer and thaw in your hand.
2. Aliquot 1-5 µl (1 pg-100 ng) of DNA to the microcentrifuge tubes. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency.
3. Incubate the cells with DNA on ice for 2 minutes.
4. After 2 minute ice incubation, heat shock the cells at 42 °C for 45 seconds.
5. Transfer the tubes to ice for 2 minutes.
6. Add 950 µl of room temperature Recovery Medium or any other medium of choice to each tube. Immediately spread 50 μl to 200 μl from each transformation on pre-warmed selection plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, plate 50 μl on an LB plate containing 100 μg/ml ampicillin. Use sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.
7. Incubate the plates overnight (12-16 hr) 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 Chemically Competent 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.