P1 Transfer of phage clones between E. coli hosts
The yield and quality of P1 phage DNA obtained from different E. coli strains depends on the genotype of the host bacterium and the specific sequence of the exogenous DNA in the recombinant. Transformation of P1 or PAC recombinants into E. coli strains that do not express Cre recombinase sometimes solves the problem of low yield or poor quality (e.g., NS3516; Sternberg et al. 1994). The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
P1 Transfer of phage clones between E. coli hosts
Principle
The yield and quality of P1 phage DNA obtained from different E. coli strains depends on the genotype of the host bacterium and the specific sequence of the exogenous DNA in the recombinant. Transformation of P1 or PAC recombinants into E. coli strains that do not express Cre recombinase sometimes solves the problem of low yield or poor quality (e.g., NS3516; Sternberg et al., 1994).
Materials and Instruments
Restriction endonuclease Closed-loop recombinant P1 DNA Electrotransformation of susceptible E. coli cells Move I. Materials For more product details, please visit Aladdin Scientific website.
Agarose gel LB agar plate SOC medium Electroporator
1. Enzyme and buffer
restriction endonuclease
2. gels
Agarose gel
3. culture medium
LB agar plates containing 25 μg/ml kanamycin
SOC medium
TB medium with 25 μg/ml kanamycin
4. Specialized equipment
Electroporator
5. Carriers and strains
Closed-loop recombinant P1 DNA
Cryopreserved electroreceptor E. coli cells (e.g. DH10B )
II. Methods
1. Dilute 2-3 μg of P1 plasmid DNA to 60 ng/μl in sterile water. set up a control sample without P1 DNA (sterile water) to be used as a parallel control during electroporation. 2.
2. Melt the transfected sensory cells on ice. Pre-cool 0.1 cm electroporation cups.
3. Mix 20 μl of cells with 1 μl of P1 DNA in the pre-cooled electroporation cup.
4. Set the electroporator to 1.8 kV, 200 Ω, 25 μF.
5. Electrocute the cells, the optimal time constant is usually about 5 ms.
6. Immediately add 0.5 ml of SOC medium preheated to 37°C to the cell suspension. Transfer the suspension to a culture tube and incubate at 37°C for 1 h with moderate shaking.
7. Spread 100 μl of cell suspension onto LB plate containing 25 μg/ml kanamycin and incubate at 37℃ overnight.
8. 10~12 colonies were placed in 11 ml of TB culture medium containing 25 μg/ml kanamycin, and incubated overnight at 37℃ with vigorous shaking.
9. Prepare P1 DNA.
10. Use different restriction endonucleases for digestion, and compare the digestion profiles of the newly extracted DNA and the original recombinant DNA by agarose gel electrophoresis.