BJ5183 感受态细胞(电转)/ BJ5183 Elect

BJ5183 Electro Competent Cell

EHC0016

 

Packing

BJ5183 Electroporation-Competent Cell  50μl/tube

pUC19 (control vector锛?0pg/μl)      10μl

Storage condition  -80鈩?for 6 month

 

Genotype

endA1 sbcBC recBC galK met thi-1 bioT hsdR (Str^R)

 

Transformation efficiency

pUC19 (control vector,10pg/ul) >10¹⁰ cfu/μg DNA

 

Transformation Protocol

 

1. Take the Electric shock cup and Cover from the storage liquid. Then inverted on clean absorbent paper for 5 minutes,until the cup and cover are dry. Inverted the Inverted cup and cover for 5 minutes, until the ethanol volatilized completed. Inserted the cup in the ice immediately, make the ice compaction. Let the the lip of a cup Higher than the ice surface 0.5cm, cover the cup then standing for 5 minutes fully cooling.

2. Take the BJ5183 competent from -80鈩?storage condition, then insert it in the ice for 5 minutes, until it is melted. Add the plasmid DNA (volume of not more than 6ul), mixed them by hand, then insert the tube in the ice, transfer the mixture to the cup by Pipette gun(200ul tips), cover the cup. Keep the tube ready to use.
a. The transformation efficiency was determined by using the control plasmid pUC19 of 1 ul at 10 pg/ul.

b. For the conjugated products, the DNA should be precipitated with ethanol and then suspended in TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) with DNA concentration not exceeding 100 ng/ul and volume not exceeding 5 ul/50 ul.

3. Start Electroporation Systems, set as C=25 μF锛孭C=200 Ω锛孷=1.8 kV (Biorad recommendation, users could also follow the manual of your Electroporation Systems), will quickly turn into the electric shock cup slot, shock quickly inserted in the ice for 2 minutesquickly.

4. Adding 700ul without antibiotics S.O.C.(room temperature) and After several times of mixing at the bottom of the shock cup with 1 ml Pipette gun, then transfer it to a 50 ml centrifugal tube (BD Falcon 50 ml conical centrifugal tube, etc.). Add S.O.C. medium to the centrifugal tube to 10 ml. At 37 C, 225 rpm was resuscitated it at 37鈩?225 rpm for 60 minutes.

 

5. Centrifuge it at 5000 rpm for one minute to collect bacteria. After suspension, Take 100-200 ml and Spread it on the S.O.C by antibiotic. Incubate overnight at 37°C.

S.O.C Medium Component

2% Tryptone

0.5% Yeast Extract

10 mM NaCl

2.5 mM KCl

10 mM MgCl2

10 mM MgSO4

20 mM glucose

PH-7.0

S.O.C. Medium is suitable for use in the final step of cell transformation to obtain

maximal transformation efficiency of E. coli (Hanahan, 1983).

 

Caution:

1. The volume of DNA should not be larger than 1/10 of the volume of the competent state.

 

2. Bubbles should be avoided when electroshock-sensitive cells are added into the shock cup. Bubbles will increase the risk of arc discharge.

 

3. When DNA is impure or contaminated by salts, ethanol, proteins and buffers, the conversion efficiency decreases sharply.

 

4. The ions in the shock cup can increase the conductivity of the solution and increase the risk of electric current and arc discharge in the solution containing cells and DNA.

 

5. If large plasmids are transformed or high transformation efficiency is desired, high purity Plasmid Extraction Kit is recommended for plasmid extraction. When the plasmid doubled, the transformation efficiency decreased by an order of magnitude.

 

6. For the transformation of conjugates, it is better to suspend the conjugates with appropriate TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) after precipitation of DNA by ethanol before transformation, so as to ensure that the concentration of DNA does not exceed 100 ng/ul. Excessive concentration of bonding products or excessive volume of bonding products will reduce the conversion efficiency and increase the risk of arc discharge.

 

7. When mixing plasmids, it should be operated gently. When absorbing competent cells, it should be avoided to exert too much force, so as to avoid excessive shear stress damaging cell membranes and reducing transformation efficiency. Conversion of high concentration plasmids or conjugates can correspondingly reduce the amount of bacteria eventually used for coating.

 

8. Electroshock-sensitive cells are best preserved below - 80°C. Over -80°C will lead to a decrease in transformation efficiency.