BL21(DE3)pLysE

BL21(DE3)pLysE Chemically Competent Cells
Storage –80° C

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Catalog No.: 961-010
Efficiency: > 107 cfu/μg pUC19 DNA
Pack Size: 1 ml (10 x 100 μL)
Price : $89

Genotype: - ompT hsdSB (rB- mB- )gal dcm (DE3)pLysE (CamR)

Suggested transformation procedure for optimal results:

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

2. Gently mix cells by lightly flicking tube. Aliquot ~50-100 μl of cells into chilled, 17 x 100 mm polypropylene tube(s), e.g., Falcon 2059. Unused cells may be refrozen, but a small drop in efficiency may result. For optimal recovery, refreeze cells in a dry ice/ ethanol bath prior to –80 °C storage.

3. Add DNA solution (< 5 μl per 50 μl cells) to cell suspension and gently swirl tube(s) for a few seconds to mix.
-If a control is desired, repeat this step with 2 μl of the provided pUC19 in a separate tube.

4. Incubate on ice for 30 minutes.

5. Place tube(s) in 42 °C water bath for ~45 seconds without shaking.

6. Replace tube(s) on ice for ~2 minutes.

7. Dilute transformation reaction(s) to 1 ml by addition of 900-950 μl SOC.
SOC Medium: 2% Tryptone, 0.5% Yeast Extract, 0.4% glucose, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 & 10 mM MgSO4.

8. Shake tube(s) ~200 rpm for 60 minutes at 37°C.

9. 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/mg pUC19 DNA) = # colonies
(colony forming units)
x 106 pg x Final volume of
transformation mix (ml)
Volume plated (μl)
pg pUC19 transformed mg

For example, if 40 colonies were obtained after transforming 200 pg of pUC19 and plating 5 ml of the final 1 ml transformation mixture, the calculated transformation efficiency would be:

40 cfu x 106 pg x 1000μl = 4 x 107 cfu/μg pUC19
200 pg pUC19   μg    5μl

*Strains are tested for expression with a control plasmid containing IL1-α