Product Introduction

The DH5 α competent cells produced by our company are prepared using a special process from Escherichia coli DH5 α strain and can be used for chemical transformation of DNA. Using pUC19 plasmid detection, the transformation efficiency can reach 108, and the transformation efficiency remains unchanged after being stored at -70 ℃ for 3-5 months.

genotype: supE44△lacU169（φ80 lacZ△M15）hsdR17 recA1 end A1 gyrA96 thi-1 relA1

Product Features

A recombinant defective inhibitory strain used for laying and cultivating plasmid and clay plates. The product of its φ 80 lacZ △ M15 gene can achieve β - complementarity with the amino terminus of β - galactosidase encoded by pUC vector, and can be used for screening blue and white spots.

Operation method(The following operations are carried out according to the standards of sterile conditions)

1. Place the competent cells on ice to melt, using 100ul of competent cells as an example in the following experiment.

2. Add the target DNA to the competent cell suspension for transformation, ensuring that the volume of the target DNA does not exceed one tenth of the volume of the competent cell suspension. Gently rotate the centrifuge tube to mix the contents and place it in an ice bath for 30 minutes.

3. Place the centrifuge tube in a 42 ℃ water bath for 60-90 seconds, then quickly transfer it to an ice bath and let it sit for 2-3 minutes, being careful not to shake the centrifuge tube.

4. Add 500ul of sterile and antibiotic free SOC or LB medium to the centrifuge tube, and shake at 37 ℃ and 180rpm for 1 hour. The purpose is to express the relevant resistance marker genes on the plasmid and revive the bacterial cells.

5. Take an appropriate amount of transformed competent cells and coat them on SOC or LB plates containing corresponding antibiotics. Invert and culture at 37 ℃ for 12-16 hours. The amount of coating can be adjusted according to specific experiments. For example, if the total amount of transformed DNA is large, about 100ul of the transformed product can be used to coat the plate; On the contrary, if the total amount of transformed DNA is small, 200-300ul of the transformed product can be coated onto the plate. Excessive bacterial liquid can inhibit bacterial growth. If there are fewer expected clones, a portion of the culture medium can be removed by centrifugation, and the bacterial cells can be suspended and coated on a plate. The remaining bacterial solution can be stored at 4 ℃. If the number of transformed colonies on the next day is too low, the remaining bacterial solution can be coated onto a new plate.

matters needing attention

1. Sensory cells should be stored at -70 ℃ and should not be repeatedly frozen or thawed, otherwise their transformation efficiency will decrease.

2. Strict aseptic operation should be carried out during the experimental process to prevent contamination by other DNA or miscellaneous bacteria, and to avoid any impact on future screening and identification.

3. During conversion, the conversion efficiency is directly proportional to the concentration of exogenous DNA within a certain range, but when too much or too large an amount of exogenous DNA is added, it can actually reduce the conversion efficiency. The volume of DNA during transformation should be less than one tenth of the volume of competent cells.

4. Calculation of conversion rate: Conversion rate=total number of bacterial colonies produced/total amount of plated DNA.

5. To prevent unsuccessful conversion experiments, some of the connected products can be retained for re conversion to minimize losses.