E.coli BL21(DE3) Competent Cells

BL21 (DE3) receptive cells produced by our company are receptive cells obtained by special processing of Escherichia coli BL21 (DE3) strain, which can be used for chemical transformation of DNA. Using pUC19 plasmid detection, the conversion efficiency can reach 107, and the conversion efficiency does not change after 6 months of storage at -70℃.

Genotype: F_ ompT hsdSB (rB_mB_) dcm gal (DE3)

Characteristics: This strain is used to efficiently express genes cloned from expression vectors containing phage T7 promoters (such as pET series). The T7 RNA polymerase is located in the DE3 region of bacteriophage λ, which is integrated on chromosome BL21. The bacterium is suitable for expressing non-toxic proteins.

Operation method: (The following steps are aseptic operation)

1. Put the receptive cells on ice to melt. The following experiment takes the 100μL receptive cells as an example.

2. Add the target DNA to be transformed into the receptive cell suspension, and pay attention to the volume of the target DNA not exceeding one-tenth of the volume of the receptive cell suspension, gently rotate the centrifuge tube to mix the contents, and place it in an ice bath for 30min.

3. Place the centrifuge tube in a water bath at 42 ° C for 60-90s, and then quickly transfer to the ice bath for 2-3min. Be careful not to shake the centrifuge tube.

4. 500μL sterile and non-resistant SOC or LB medium was added into the centrifuge tube and cultured for 1h by oscillating at 180rpm at 37℃. The aim is to make the relative resistance marker genes expressed on the plasmid, so as to revive the bacteria.

5. Appropriate amount of transformed receptive cells were coated with SOC or LB plates containing corresponding antibiotics and cultured invert at 37℃ for 12-16h. The amount of coating can be adjusted according to the specific experiment, such as the total amount of converted DNA is more, the conversion product coating plate of about 100μL is preferable; Conversely, if the total amount of converted DNA is small, 200-300μL of conversion product coating is preferable. Excessive bacterial fluid can inhibit bacterial growth. If few clones are expected, part of the culture solution can be removed by centrifugation, and the bacteria can be suspended and coated on a plate. The remaining bacterial solution can be stored at 4℃, and if the number of transformed colonies is too low the next day, the remaining bacterial solution can be coated with a new plate.

Note:

1. The receptive cells should be stored at -70℃, can not be frozen and thawed repeatedly, otherwise its conversion efficiency will be reduced.

2. The experiment should be strictly aseptic operation, to prevent the contamination of other DNA or miscellaneous bacteria, to avoid the impact on future screening and identification.

3. During conversion, the conversion efficiency is proportional to the concentration of foreign DNA within a certain range, but when the amount of foreign DNA added is too large or the volume is too large, the conversion efficiency will be reduced. At the time of transformation, the DNA volume is less than one-tenth of the volume of the receptive cell.

4. Calculation of conversion rate: conversion rate = total number of colonies generated/total amount of pavement DNA.

5, In order to prevent the conversion experiment from being unsuccessful, some of the connected products can be retained to re-transform and minimize the loss.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.

Remark：These protocols are for reference only. Solarbio does not independently validate these methods.

Note：

1. The products are all for scientific research use only. Do not use it for medical, clinical diagnosis or treatment, food and cosmetics, etc. Do not store them in ordinary residential areas.

2. For your safety and health, please wear laboratory clothes, disposable gloves and masks.

3. The experimental results may be affected by many factors, after-sale service is limited to the product itself and does not involve other compensation.