HB101

HB101 Electroporation Competent Cells
Storage –80 C

Click here to download protocol in PDF format (print-friendly)

Catalog #: 961-009
Efficiency: ≥ 109 cfu/μg pUC19 DNA
Pack Size: 0.5 ml (5 x 100 μL)
Price : $79

Genotype: -, hsdS20(rB-, mB-), xyl5, λ -, recA13, galK2, ara14, supE44, lacY1, proA2, rpsL20(str r), leuB6, mtl-1, thi-1

Lot Efficiency: This lot of electroporation competent cells was tested with an EquiBio Easyject Optima electroporator using a 0.1 cm cuvette. Using settings recommended by the manufacturer and protocol as described below, actual pulse times were >4.6 ms and transformation efficiencies >109 cfu /μg pUC19 DNA.

Suggested transformation procedure for optimal results:

1. Pre-chill electroporation cuvettes, electroporation chamber (if applicable), and microcentrifuge tubes on ice.

2. Remove cells from -80° C and let thaw on ice.

3. Place 40-50 μl of the competent cells into a chilled microcentrifuge tube. Add 1-5 μl of sample DNA to cells. Thoroughly mix by gently pipetting and incubate on ice for approximately 1 minute.

Note: For optimal results, sample DNA should be in sterile H2O or low ionic strength buffer such as TE. If a control is desired, repeat this step with 2 μl of the provided pUC19 in a separate tube. Refreeze any unused cells and store at -80° C.

4. Transfer cell mixture into a pre-chilled cuvette and pulse using settings recommended by manufacturer of electroporator. As a general guideline, maximum transformation efficiency is normally attained using cuvettes with a 0.1 cm gap with an applied voltage of ~1800 (field strength of ~18 kV/cm).

5. Immediately dilute pulsed cells to 1 ml with SOC medium and transfer to a sterile culture tube.
SOC: 2% Tryptone, 0.5% Yeast Extract, 0.4% glucose, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 & 10mM MgSO4.

6. Gently shake culture tube ~200 rpm for 60 minutes at 37°C.

7. Plate by spreading 5-200 μl of cell transformation mixture on LB agar plates containing appropriate antibiotic and incubate overnight at 37°C.
-When performing the pUC19 control transformation (pUC19 supplied as 10 pg/μl), plate 5 μl of the transformation mixture on a LB agar plate containing 100 μg/ml ampicillin. To facilitate cell spreading, place a pool of SOC (100μl) onto surface of plate prior to addition of transformation mixture.

Transformation Efficiency Calculation for Control DNA

Transformation Efficiency (cfu/µg pUC19 DNA) = # colonies
(colony forming units)
x 106pg x Final volume of
transformation mix (ml)
Volume plated (μl)
pg pUC19 transformed µg

 

For example, if 30 colonies were obtained after transforming 20 pg of pUC19 and plating 5 µl of the final 1 ml transformation mixture, the calculated transformation efficiency would be:

200 cfu x 106 pg x 1000μl = 2 x 109 cfu/μg pUC19
20 pg pUC19 μg 5μl