Plant Molecular Biology (S9)
The chloroplast genome of higher plants is relatively small and simple. This genome contains about 120 genes which code for chloroplast proteins. The organelle's genome also contains ribosomal RNA genes which code for the RNA components of the chloroplast ribosome. In this program, students identify the ribosomal RNA genes in the chloroplast genome of spinach. Students first isolate chloroplasts from fresh spinach and then prepare DNA from the isolated organelle. The DNA is digested with EcoR1, electrophoresed, and the separated fragments transferred to a nylon membrane. The DNA fragments containing the ribosomal genes are then detected by hybridization using a biotin-labeled probe made from a plasmid that contains the ribosomal gene sequences. Fresh spinach, microscopes, ethyl alcohol, a water bath incubator that will maintain a temperature of 60-65°C, and a centrifuge that can be operated at a force of at least 3000 x g are needed for the program. A microcentrifuge is also desirable, but not absolutely necessary.
A typical laboratory schedule for this program is given below where each lab session is approximately 3 hours.
Lab Session 1
Students isolate chloroplast from spinach leaves and isolate DNA from the chloroplast by a unique procedure that yields high quality DNA without the use of toxic organic solvents.
Lab Session 2
The purified DNA is digested with the restriction endonuclease EcoR1, and then electrophoresed on agarose gels.
Lab Sessions 3-5
The DNA fragments in the gels are transferred to nylon membranes using the transfer devices provided with the program. The fragments containing the genes for ribosomal RNA are then detected by hybridization using a biotin-labeled probe prepared from a plasmid containing the ribosomal gene sequences. Typical results are shown.
Left: Marker DNA and chloroplast DNA samples were electrophoresed on an agarose gel and the gel was stained. Right: The separated DNA fragments on the gel were transferred to a nylon membrane and the DNA containing the chloroplast ribosomal RNA genes was identified by hybridization analysis.