HB101 Chemically Competent Cells
HB101 Chemically Competent Cells Storage –80° C
|Catalog No.||Efficiency||Pack Size||Price|
|961-013||≥ 108 cfu/μg pUC19 DNA||1 ml (5 x 200 μL)||$89|
|961-025||≥ 108 cfu/μg pUC19 DNA||1 ml (20 x 50 μL)||$99|
Genotype: F-, hsdS20(rB-, mB-), xyl5, λ-, recA13, galK2, ara14, supE44, lacY1, proA2, rpsL20(strr), leuB6, mtl-1, thi-1
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
(cfu/mg pUC19 DNA)
(colony forming units)
|x||106 pg||x||Final volume oftransformation mix (ml)Volume plated (μl)|
pg pUC19 transformed
For example, if 40 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:
|40 cfu||x||106 pg||x||1000μl||=||4 x 108 cfu/μg pUC19|
|20 pg pUC19||μg||5μl|