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TaKaRa E.coli DH5ααααα Electro-Cells

TAKARA BIO INC.

Cat.# 9027

Contents:

Specification:

TaKaRa E.coli DH5α Electro-Cells .................. 10 vials x 50 µlpUC19 plasmid (10 pg / µl) .................................. 1 vial x 10 µlSOC medium• ..................................................... 10 vials x 1ml

• SOC medium: 2% Bacto tryptone

0.5% Bacto yeast extract

10 mM NaCl

2.5 mM KCl

20 mM MgSO4•MgCl2* (each 10 mM)

20 mM Glucose*

TaKaRa Electro-Cells are specially prepared by Takara to be best appropri-ate for electroporation method. Electroporation method is used to transferDNA into a cell by breaking cytoplasmic membrane by high voltage pulse.As this electro-cells offers high transformation efficiency and goodreproducivility, it is especially useful in transferring small amount of sampleinto a E.coli in least time.TaKaRa E. coli DH5α Electro-Cells is a host for Blue / White screeningutilizing the activity of β-galactosidase ( α-complementation ) in combinationuse of pUC vectors. As this strain does not carry laclq, basically IPTG is notneeded. Therefore, TaKaRa E. coli DH5a Electro-Cells allows easy selec-tion of recombinant DNA with X-Gal when constructing gene library orsubcloning recombinant plasmid.

Transformation into a plasmid vector

1) Thaw 50 µl of TaKaRa E.coli DH5α Electro-Cells in an ice bath justbefore use.

2) Add 1-2 µl of DNA solution* into the thawed cell suspension.*When sample DNA solution contains salt, dilute with TE buffer ordistilled sterilized water.Or desalting by ethanol precipitation is recom-mended. The ethanol precipitation shall be performed, when the samp-le DNA was prepared with TaKaRa DNA Ligation Kit (Ver.1and Ver. 2).

3) Transfer the mixture of cells and DNA to a cold 0.1 cm electroporationcuvette.

4) After applying pulse*, immediately add 1 ml of SOC medium (precooledin an ice bath).*Takara uses a BIO-RAD's gene pulser. The electrical conditions are25 µF, 200 Ω under the voltage condition in the supplied lot card.

5) Incubte by shaking (160-250 rpm) for 1 hour at 37°C.6) Plate on selective media.7) Incubate overnight at 37°C.

1) Place a vial of electro-cells in a dry ice / EtOH bath immediatelyupon removal from -80°C freezer. Keep cells in bath until you are readyto proceed.

2) Freeze the unused portion of cells in dry ice / EtOH bath and returnthem to the -80°C freezer.

3) When using 50 µl of electro-cells, apply high-purified sample DNAin less than 10 ng. If not, transformation efficiency might decrease.

4) When changing an experiment scale, optimum condition should beconsidered.

5) When transferring high molecular weight DNA (>7 kb), transformationefficiency may decrease.

6) Use TE buffer for sample DNA preparation. High salt concentration insample DNA solution may decrease transformation efficiency.

7) L-broth or ψ b-broth can be used instead of SOC medium. In this case,lower efficiency might be obtained.

Protocols:

Please read before proceeding:

v.02.04

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TaKaRa E.coli DH5ααααα Electro-Cells

TAKARA BIO INC.

• L-broth : Ingredient per liter waterBacto tryptone .................10 gBacto yeast extract .......... 5 gNaCl ................................... 5 g

Adjust to around pH7.5 with 1N NaOH and autoclave.

• ψ b-broth: Ingredient per liter waterBacto tryptone ................ 20 gBacto yeast extract ............ 5 gMgSO4•7H2O....................... 5 g

Adjust to around pH7.5 with 1N KOH and autoclave.

8) When diluting, use SOC medium which has been added in the step4) of Protocols.

9) When adding X-Gal to medium, follow the procedure described asbelow.• Add 20 mg / ml dimethylhormeamide of X-Gal into 200 µl / 100 ml agar medium.

10) DH5α can be used for the replication of M13mp vectors.But the strain can not form plaques, as it does not carry F factor.

10 pg of pUC19 was transformed and transformants were selected by Amp+

selective media plating.Transformation efficiency : 1 x 109 transformants / µg pUC19

Blue colony appeared when pUC19 DNA was transformed, then plating thetransformed cells on a L-agar medium containing 100 µg / ml of ampicilinand 40 µg / ml of X-Gal.

E. coli DH5α: F-, φ80dlacZ∆M15, ∆(lacZYA-argF)U169, deoR, recA1,endA1, hsdR17(rk

-, mk+), phoA, supE44, λ-, thi-1, gyrA96,

relA1

>1x 1010 bacteria/ml

-80°CNote: If it is not stored at -80°C, transformation efficiency may decrease.

1. Dower, W.J., Miller, J.F. and Ragsdale, C.W. (1988) Nucl.Acids Res.,16, 6127.

2. Bottger, E.C. (1988) Biotechniques, 6, 878.

Transformation efficiency:

ααααα-complementation of β β β β β-galactosidase:

Genotype:

Cell density:

Storage:

Reference:

NOTE: For research use only.Not for use in diagnostic or therapeutic procedures.