PX5α MCR

PX5α MCR
Chemically Competent Cells                                                Storage –80° C

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

 

Catalog No. Efficiency Pack Size Price
961-005 > 108 cfu/mg pUC19 DNA 1 ml (5 x 200 μL)  $89

 

Genotype: deoR, endA1, recA1, relA1, gyrA96, hsdR17(rkmk+), supE44, thi-1, Δ(lacZYA-argFU169), φ80δlacZΔM15, mcrA Δ(mrr-hsdRMS-mcrBC)-, λ -

 

Suggested transformation procedure for optimal results:span>

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 ml 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 ml 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 (100 μl) 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 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