In this protocol, when the exogenous DNA is larger than 200~300 nucleotides, it can be identified by gel electrophoresis analysis of the single-stranded DNA released into the surrounding medium after infecting the bacteria with the recombinant M13 phage clone. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Analysis of recombinant M13 phage clones
Principle
In this scheme, when the exogenous DNA is larger than 200-300 nucleotides, it can be identified by gel electrophoresis analysis of the single-stranded DNA released into the surrounding medium after infection of the bacteria with the recombinant M13 phage clone.
Materials and Instruments
Recombinant M13 phage single-stranded DNA M13 phage recombinant phage M13 phage non-recombinant vector phage E. coli F' strain Move I. Materials For more product details, please visit Aladdin Scientific website.
SDS SSC Sucrose Gel Spiking Buffer
Agarose gel Water bath
1. Buffers and solutions
SDS ( 2%, m/V)
20 X SSC
Sucrose Gel Spiking Buffer
2. Gel
Agarose gel (0.7%) suspended in 0.5 X TBE, containing 0.5 μg/ml ethidium bromide.
3. nucleic acids and oligonucleotides
Recombinant M13 phage single-stranded DNA
4. Specialized equipment
Water bath, set to 65°C
5. Carriers and strains
Recombinant phage spots of M13 phage in the upper agarose layer
Well-isolated M13 phage non-recombinant vector phage spots in the upper agarose layer
E. coli F' strain
II. METHODS
1. Take a single phage spot and grow it in a suitable F' host as described in the protocol "M13 phage liquid culture" to prepare a prototype of the putative recombinant phage.
2. Remove 20 μl of each supernatant with a micropipette with a sterile tip into a new microcentrifuge tube and store the remaining supernatant at 4℃ for later use.
3. Add 1 μl of 2% SDS to each 20 μl of supernatant, tap the wall of the tube to mix the contents, and incubate at 65℃ for 5 min.
4. Add 5 μl of sucrose spiking buffer to each tube, tap the mixed contents again, and analyze by 0.7% agarose gel electrophoresis at 5 V/cm. The identified single-stranded DNA of M13 recombinants with exogenous sequences of known size was used as a positive control.
5. Bromophenol blue was photographed under ultraviolet light after migration to the ends of the gel.
6. Compare the electrophoretic mobility of single-stranded DNA released by the putative recombinants with that released by non-recombinant phages used as controls.
7. If desired, transfer the single-stranded DNA on the gel to a nitrocellulose or nylon membrane and hybridize with an appropriate radiolabeled probe to confirm the presence of exogenous DNA. The gel is first soaked in 10 times the volume of 20 X SSC for 45 min, and then the DNA is transferred directly to the membrane. 