Amplify and purify the stretch of DNA of interest: Use an amplification protocol that has already been evaluated and optimized for the PCR reagents available and specific to the laboratory. Prepare the PCR reagents such as primers and if phosphorylate these if blunt-ended ligation is the cloning method of choice. For the PCR, generally 20 to 25 cycles of amplification with hybridization for a minute at 50 degrees Celsius are sufficient to produce complete products. Take 5 to 10 microliters of the PCR product and run it out on an agarose gel to visualize and confirm the size of the DNA. Recover and purify the PCR products by the basic techniques of phenol-chloroform extraction and ethanol precipitation.
Enzymatically digest and repurify the DNA fragments: The user should already know what restriction enzyme sites are present within the amplified DNA prior to starting any work, and these sites will be used in setting up a digestion to 'introduce' the naked sites to the fragments. Use approximately half of the total amplified volume for this and after digestion, repurify the fragments by gel and phenol-chloroform extraction or other methods.
DNA ligation and cellular transfection: Carry out a DNA ligation experiment to place the restricted fragment within a vector that can be transfected into a bacterial cell. The cells will transcribe the new recombinant gene and produce large quantities of it. Grow up these bacteria and carry out subcloning experiments to allow the selection of potential transformants.
Analyze the subclones to confirm the modified gene: After mini-, midi-, or maxiprepping the plasmid (expression vectors), repeat the restriction enzyme digestion and gel visualization process, and sequence the amplified fragment to verify that no mutations are present.