General DNA Cloning

The basic steps involved in producing recombinant DNA are outlined in Figure 1.

  1. Plan the cloning strategy. This includes selecting a method for obtaining the DNA fragment to be cloned, selecting a vector into which the DNA fragment will be incorporated, and selecting the bacterial host that will be used to produce the recombinant DNA.
  2. Isolate the vector and the DNA fragment to be cloned. For the vector this may involve growing an appropriate bacterial strain carrying the vector, and isolating the vector DNA. The method used to obtain the DNA for cloning depends on where it currently resides (in a plasmid, in genomic DNA, in mRNA etc.).
  3. Prepare the vector and the target DNA for ligation. The DNA that will be cloned needs to be an appropriate size for ligation (size range depends on the vector being used) and the vector needs to be opened up (made linear) so that the insert can be ligated to it. This could involve using restriction enzymes or mechanical methods to breakup the target DNA. Vectors are usually opened up using restriction enzymes.
  4. Ligate the digested vector with the DNA fragment(s) of interest.
  5. Screen the new plasmids to find the desired recombinant plasmid(s). This involves transforming host bacteria with the ligation mixture and using selection methods to isolate single bacteria carrying the newly created plasmids. Plasmids are then re-isolated and checked for correct structure.
Figure 1. Cloning Steps
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