Product Specification
TG1 Electroporation-Competent Cell 50μl/tube
pUC19 (control vector，10pg/μl): 10μl
Storage conditions (shelf life): -80℃（6 months）
Genotype
[F´ traD36 proAB lacIqZΔM15] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK–mK–)
Product Description
TG1 electroporation competent cells can only be used for electroporation transformation and cannot be used for heat shock transformation. TG1 was derived from E. coli K-12 strain, which is the fastest growing clone of E. coli strain, and cloned at 37 ° C for 7 h on the plate. The main phage display strain can also be used for the construction of common plasmids. The presence of lacIqZΔM15 can be used for experiments such as blue-white spot screening; but it does not contain nuclease endA1 mutation, and the nuclease content in the body is high. It is recommended to use when extracting plasmid. The deproteinized solution in the plasmid extraction kit is used to remove a large amount of nuclease from the bacteria. The TG1 electroporation competent cells were suitable for the construction of phage display library. The pUC19 plasmid was used to detect the transformation efficiency of >0.5×1010 cfu/μg DNA.
Operation Method
1. The 0.1 cm electric shock cup and lid are removed from the stock solution and placed on clean absorbent paper for 5 minutes. Allow it to drain and set it for 5 minutes to allow the ethanol to fully evaporate. After the ethanol is evaporated, immediately insert it into the ice, and compress the ice surface. The top of the electrode cup should be 0.5 cm away from the ice surface and allow it to stay in the ice for 5 minutes to fully cool down.
2. Insert the TG1 shock-competent cells stored at -80 °C into the ice for 5 minutes. After it is melted, add the DNA of interest (plasmid or ligation product) and gently mix the bottom of the EP tube by hand to avoid air bubbles and immediately insert into the ice.
A. Determination of transformation efficiency using 1 μl 10pg / μl of the control plasmid pUC19;
B. For the ligation product, precipitate the DNA with ethanol and resuspend by adding the appropriate amount of TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA). The DNA concentration does not exceed 100 ng/μl and the volume does not exceed 5 μl/50 μl.
3. Using a 200 μl tip (using a knife to remove the 0.5 cm tip), quickly transfer the competent-DNA mixture to the shock cup to avoid air bubbles and cover the lid.
4. Start the electro-rotator and set the parameters: C=25 μF, PC=200 Ω, V=1.8 kV (this is the recommended parameter of the BioRad motor, and can also be operated according to the parameters recommended by the instrument used). Quickly insert the electric shock cup into the electric transfer slot, and the electric shock is quickly inserted into the ice.
5. Remove the electric shock cup from the ice after 2 minutes and let it stand at room temperature. Add 1 ml of sterile S.O.C. medium (room temperature) without antibiotics, and mix the bottom of the electric shock cup with a 1 ml micropipette for several times. Transfer to a 50 ml centrifuge tube (BD Falcon 50 ml conical centrifuge tube, etc.) and add S.O.C. medium to the centrifuge tube to 10 ml. Place at 45 degrees on a shaker and resuscitate at 37 ° C for 225 rpm for 60 minutes.
6. Centrifuge at 5000 rpm for one minute, resuspend and apply 100-200 μl to the SOC plate containing the corresponding antibiotics. (The amount of bacteria is large. If all the plates are coated, please use 2-5 diameter culture dishes.) . Place the plates upside down in a 37 ° C incubator for 13-17 hours.
Attention
1. When adding DNA, the volume should not be greater than 1/10 of the competent volume.
2. Electric shock cells should be added to the electric shock cup to avoid bubbles, which will increase the risk of arc discharge.
3. When the DNA is impure or contaminated with salt, ethanol, protein and buffer, the conversion efficiency drops dramatically.
4. Ions in the electric shock cup increase the conductance of the solution and increase the risk of current and arc discharge in solutions containing cells and DNA.
5. If you are transforming a large plasmid or want to achieve higher conversion efficiency, it is recommended to extract the plasmid using the high purity plasmid extraction kit. The plasmid is doubled and the transformation efficiency is reduced by an order of magnitude.
6. For ligation of the ligation product, it is best to precipitate the ethanol before precipitation and resuspend the product with an appropriate amount of TE buffer (10 mM Tris HCl, pH 7.5; 1 mM EDTA) to ensure that the DNA concentration does not exceed 100 ng/μl. Excessively high concentrations of the linking product or excessive volume of the linked product reduce conversion efficiency and increase the risk of arc discharge.
7. When mixing the plasmid, it should be gently operated. Avoid excessive force when aspirating the competent cells, so as to avoid damage to the cell membrane and reduce the transformation efficiency. Transformation of a high concentration of plasmid or ligation product can correspondingly reduce the amount of bacteria ultimately used for plating.
8. The electroporation competent cells are best kept below -80 °C, and stored at temperature above -80 °C will result in a decrease in conversion efficiency.
