Product Specification
BL21 (DE3)：110ul/tube
pUC19 (control vector，10pg/μl): 10μl
storage condition (Guarantee period)： -80℃（6 months）
Genotype
F–ompT hsdS(rB– mB–) dcm+ Tetr gal(DE3) endA Hte [argU proL Camr] [argU ileY leuW Strep/Specr]
Product Description
BL21(DE3) receptor cell is a kind of receptor cell produced by Escherichia coli BL21(DE3) after special treatment, which can be used for chemical transformation of plasmid. PUC19 plasmid were used to detect, conversion efficiency can be as high as 108 CFU/ug, storing at -70℃ for a few months would not change the efficiency of the conversion.
E. coli BL21(DE3) is the most basic prokaryotic protein expression strain because of its rapid growth and high expression. The defective type of protease Lon and OmpT can effectively reduce the degradation of target proteins. Suitable for iptg-induced T7 promoter vector, commonly used pET series carrier; Expression of non-toxic protein, codon preference.
Operation method
1. Take BL21(DE3) receptor cells and place them in an ice bath. If necessary, the newly melted cell suspension can be divided into a sterile pre-cooled centrifuge tube and placed in an ice bath.
Note: the recommended dosage of a transgenic BL21(DE3) receptor cell is 50-100μl. It can be used separately according to the actual situation. It should be noted that the volume of DNA used should not exceed one tenth of the suspended fluid volume of the cells in the receptor state. The following experiment took 100 cells in the receptor state of l BL21(DE3) as an example.
2. Add the target DNA to the BL21(DE3) receptor cell suspension (the 100 igc cells can be saturated with 1 ng super-helical plasmid DNA), flick and mix well, and leave in the ice bath for 30 minutes.
3. Place the centrifuge tube at 42 ℃ water bath in 60-90 seconds, and then quickly transfer the centrifuge tube to the ice bath, to let the BL21 (DE3) cells cool for 2-3 minutes, do not shake the centrifuge tube.
Note: this procedure can also be carried out at room temperature with the centrifuge tube at room temperature. The time is approximated. If the room temperature is low, it can be extended to about 8-15 minutes. Conditions allow recommended 42 ℃ heat shock method.
4. To each centrifuge tube add 900 ul sterile SOC or LB culture medium (including antibiotics), mix it and place it on 37 ℃ shaking bed for 45 minutes (150 RPM), the purpose is to make the related resistance mark in the plasmid gene expression, to allow the bacteria recovery.
5. Mix the contents in the centrifuge tube, place 100 ul of transformed BL21 (DE3) cells on the SOB or LB solid AGAR culture medium containing corresponding antibiotics on the tablet, use a sterile bend glass rod or glass bead to spread out the cell. Put tablet at room temperature until the liquid is absorbed completely, then inverse the tablet, 37 ℃ for 12-16 hours.
Note: coating dosage can be adjusted according to specific experiment. If the total amount of transforming plasmid is more, less transformation product coating plate is preferable. On the contrary, if the total amount of transformed plasmid is small, it is advisable to use 200-300 co-coated tablets of the transformation product. For the conjunctive solution, most of the culture medium supernatant can be removed by centrifugation (4000 RPM, 2 min). After blowing the suspended bacteria, it can be coated on a flat plate.
6.The remaining bacteria liquid can be placed in 4 ℃ refrigerator, if the number of colonies is too little, can put the rest of the bacteria liquid on a new tablet.
Attention
1. BL21 (DE3) cells should be stored at - 70 ℃, no repeated freezing and thawing and long time storage, so as not to reduce the conversion efficiency of cells.
2. The transformation shall be carried out according to the corresponding temperature and aseptic conditions.
3. In order to prevent the unsuccessful transformation experiment, part of the connected reaction fluid can be retained to re-convert and minimize the loss.
4. The volume of the junction product or plasmid used for transformation shall not exceed 15% of the volume of the receptor cell.
5. After the plasmid exceeds 7.5kb, the conversion efficiency will decrease with the increase of size. When the size of plasmid exceeds 10kb, plasmid loss may also occur in the process of E. coli proliferation.
6. The amount of plasmid used for transformation is the best between 0.1pg and 50ng. When the amount of plasmid exceeds 50ng, the number of monoclonal cells on the plate will increase, but the conversion efficiency will decrease.
7. 42 ℃ hot shot time and the thickness of the centrifuge tube, bacteria liquid is directly related to the size of the volume, general advice hot hit for 60-90 seconds, hot shot time should not be more than 90 seconds, or efficiency will be decreased rapidly;
