1. Bacteria Transformation
Bacteria are useful instruments for gene study because they can be induced (coerced) into taking up foreign DNA (transformation) for later expression.

2. Adding DNA to E-Coli
Most techniques for cloning make use of bacteria (E. coli) as they have small circular plasmids that are receptive for insertion of foreign DNA.
Plasmid is isolated from the bacteria
the plasmid useful for may already be prepared with an insertion site (restriction site) and an enzyme that infers antibiotic resistance (ampR)
restriction enzymes are used to isolate the gene of interest and to open the plasmid
DNA is attached to the plasmid, at the sticky ends using DNA ligase
DNA containing the gene of interest is mixed with the open plasmids so that the sticky ends can attach
DNA ligase is added after to permanently join the segments
now called the plasmid cloning vector

3. Confirmation of Transformation
Plasmid DNA with the gene inserted is now known as recombinant DNA
Plasmid is inserted back into the bacteria via transformation and grown on special agar containing an antibiotic or color marker
Competent (transformed bacteria) are harvested and grown on new plates
The bacteria produces clones of itself through binary fission
The products can then be harvested

4. YAC Yeast cells also make good cloning vectors
contain a larger plasmid
are single celled eukaryotic organisms
can incorporate YACs (yeast artificial chromosomes) into the organism
reproduce by mitosis and contain their own promoter regions
allow for even larger DNA pieces to be added
DNA can be incorporated into animal cells through the process of electroporation
electrical impulses that poke holes in the plasma membrane
YAC

5. Knock-out genes Genes are inserted into a eukaryotic egg
Similar sequence as known gene
Contains a marker for study
Allows the researcher to see affect that the absent product has on the organism

6. DNA Libraries
Cloned DNA can be stored in DNA libraries for future use and study
can be packaged in bacteriophages (phage vector) for quick insertion and cloning bacteria plasmids
a complete set of phage clones is called a genomic library
an advantage is that larger DNA particles can be incorporated in the phage vector than in the plasmid DNA
DNA libraries can also be created from the mRNA transcribed from the genes - called cDNA libraries
reverse transcriptase is used to make the complimentary DNA strand
the cDNA is then inserted into a vector and stored
an advantage is the cDNA contains only exons as the mRNA is already modified from the original transcript