TaKaRa E.coli DH5 Electro-Cells · TaKaRa E. coli DH5α Electro-Cells is a host for Blue / White...
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1 TaKaRa E.coli DH5α Electro-Cells TAKARA BIO INC. URL:http://www.takara-bio.com v.050711 Cat.# 9027 Contents: Specification: TaKaRa E.coli DH5α Electro-Cells .................. 10 vials x 50 μl pUC19 plasmid (10 pg / μl) .................................. 1 vial x 10 μl SOC 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 transfer DNA into a cell by breaking cytoplasmic membrane by high voltage pulse. As this electro-cells offers high transformation efficiency and good reproducivility, it is especially useful in transferring small amount of sample into a E.coli in least time. TaKaRa E. coli DH5α Electro-Cells is a host for Blue / White screening utilizing the activity of β-galactosidase ( α-complementation ) in combination use of pUC vectors. As this strain does not carry lacl q , basically IPTG is not needed. Therefore, TaKaRa E. coli DH5a Electro-Cells allows easy selec- tion of recombinant DNA with X-Gal when constructing gene library or subcloning recombinant plasmid. Transformation into a plasmid vector 1) Thaw 50 μl of TaKaRa E.coli DH5α Electro-Cells in an ice bath just before use. 2) Add 1-2 μl of DNA solution* into the thawed cell suspension. *When sample DNA solution contains salt, dilute with TE buffer or distilled sterilized water.Or desalting by ethanol precipitation is recommended. The ethanol precipitation shall be performed, when the sample 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 electroporation cuvette. 4) After applying pulse**, immediately add 1 ml of SOC medium (pre cooled in an ice bath). **Takara uses a BIO-RAD's gene pulser. The electrical conditions are 25 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.*** *** ≤100 μl is recommended for plating on dish with ∅9cm. 7) Incubate overnight at 37°C. 1) Place a vial of electro-cells in a dry ice / EtOH bath immediately upon removal from -80°C freezer. Keep cells in bath until you are ready to proceed. 2) Freeze the unused portion of cells in dry ice / EtOH bath and return them to the -80°C freezer. 3) When using 50 μl of electro-cells, apply high-purified sample DNA in less than 10 ng. If not, transformation efficiency might decrease. 4) When changing an experiment scale, optimum condition should be considered. 5) When transferring high molecular weight DNA (>7 kb), transformation efficiency may decrease. 6) Use TE buffer for sample DNA preparation. High salt concentration in sample DNA solution may decrease transformation efficiency. Protocols: Please read before proceeding:
Transcript of TaKaRa E.coli DH5 Electro-Cells · TaKaRa E. coli DH5α Electro-Cells is a host for Blue / White...
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TaKaRa E.coli DH5α Electro-Cells
TAKARA BIO INC.URL:http://www.takara-bio.com
v.050711
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 or distilled sterilized water.Or desalting by ethanol precipitation is
recommended. The ethanol precipitation shall be performed, when the sample 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 electroporation
cuvette.4) After applying pulse**, immediately add 1 ml of SOC medium (pre cooled in 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.*** *** ≤100 µl is recommended for plating on dish with ∅9cm.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.
Protocols:
Please read before proceeding:
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TaKaRa E.coli DH5α Electro-Cells
TAKARA BIO INC. URL:http://www.takara-bio.com
Cat.# 9027v.050711
7) L-broth or ψ b-broth can be used instead of SOC medium. In this case,lower efficiency might be obtained.
• 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.
