Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

XLDNA V2 Electrocompetent Cells

Preparation for Transformation

Large-insert DNA cloning and BAC library construction demand the highest transformation efficiency and recovery of the largest clones possible. Sigma’s XLDNA V2 Electrocompetent cells were developed exclusively for this purpose. We also recommend use of the Cell Porator and Voltage Booster System from Whatman Biometra. Optimal settings for electroporation are listed below:

Table: Electroporation Conditions
Optimal Setting (Recommended) Alternate Conditions (May give lower efficiencies)
Cuvette gap: 1.5 mm Cuvette gap: 1.0 mm
Voltage : 358 V Voltage : 1800 V
Capacitance: 330 µF Capacitance: 25 µF
Impedance: Low ohms Impedance: 200 ohms
Charge rate: Fast  
Voltage Booster  
Resistance: 4000 ohms  

Optional transformation control reactions include electroporation with 1 ul (10 pg) of supercoiled pKanR DNA.

To ensure successful transformation results, the following precautions must be taken:

  • For best results, the BAC ligation reaction must NOT be purified or heat treated.
  • The DNA sample to be used for electroporation must be dissolved in water or a buffer with low ionic strength, such as TE. The presence of salt in the electroporation sample leads to arcing at high voltage, resulting in the loss of the cells and DNA.
  • Microcentrifuge tubes and electroporation cuvettes must be thoroughly pre-chilled on ice before use. Optimal results are obtained with the electroporator and cuvettes from Whatman Biometra (Cat.# 11609013 and #11608031, respectively). Successful transformation also can be achieved with alternate systems, although efficiency may be lower (see Table 2).
    The cells must be completely thawed on ice before use.
  • For highest transformation efficiency, use the provided Recovery Medium to resuspend the cells after electroporation. Use of TB or SOC will result in lower transformation efficiencies.
  • Use YT agar plus appropriate antibiotic for plating cells. Colony size will be small or variable on LB agar plates. YT Agar is used to maximize colony size. Cells may be plated on LB or other common media—colonies will be noticeably smaller upon comparison but adequate for the vast majority of intents and purposes.

Transformation Protocol

  1. Aliquot 1 ml of Recovery Medium into 17 mm x 100 mm sterile culture tubes at room temperature (one tube for each transformation reaction). Transformation efficiency may decrease with the use of other media.
  2. Place electroporation cuvettes and microcentrifuge tubes on ice (one cuvette and one tube for each transformation reaction).
  3. Remove cells from the -80°C freezer and place on wet ice until they thaw completely (10-20 minutes).
  4. When cells are thawed, mix them by tapping gently. Add 20 μl of cells to the chilled microcentrifuge tube on ice.
  5. Add 1 μl of the BAC ligation reaction directly to the 20 μl of cells on ice. Do NOT heat inactivate the ligation reaction. (Heat-inactivating the ligation reaction will reduce the quality of BAC cloning.) Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells. Using more than 1 μl of ligation mix may cause electrical arcing during electroporation.
    For ligation reactions using other commercial kits, please refer to the manufacturer’s instructions.
  6. Carefully pipet the cell/DNA mixture into a chilled electroporation cuvette without introducing bubbles. Electroporate according to the conditions recommended on p.4.
  7. After electroporation, quickly transfer the cells into the Recovery Medium in the culture tube at room temperature.
  8. Place the tube in a shaking incubator at 250 rpm for 1 hour at 37 °C.
  9. Spread up to 100 μl of transformed cells on YT agar plates containing the appropriate antibiotic.
  10. Incubate the plates overnight at 37 °C.
  11. Transformed clones can be further grown in TB or in any other rich culture medium.

DNA Isolation

Transformants are grown in TB medium. Stable inserts of 10-40 kb can be grown overnight with shaking at 37 °C in the presence of 1X Arabinose Induction Solution. DNA minipreps can be performed by standard methods.

For BACs and unstable smaller inserts, it may be necessary to grow the cultures without induction to an OD600 of 0.2-0.3. To reach this OD, it is convenient to grow the cultures overnight at 37 °C without shaking. The following morning, dilute the cultures 2-10 fold, and grow at 37 °C with shaking at 225 rpm for 30 minutes. For each ml of culture, add 1 μl of 1000 X Arabinose Induction Solution. Continue growth for 2-3 hours at 37 °C with shaking at 225 rpm.

Prepare DNA minipreps according to standard protocols.

Materials

     
Related Links