Plasmid Extraction - Middle Tennessee State University：质粒提取-田纳西中部州立大学.doc

Chloroplast Genome Project – Week 1 In plant cells, DNA can be found in three organelles – nuclei, chloroplasts, and mitochondria. The chloroplast is the site of photosynthesis in plant cells. It’s genome and its process of gene expression is very similar to those found in bacteria. The chloroplast genome can be imagined as a circular piece of DNA about 120,000 – 140,000 bases in size with approximately 100 genes. This diagram represents the corn chloroplast genome which was completed in 1995. You and your genetics class colleagues are attempting to sequence the chloroplast genome of tall fescue (Festuca arundinacea). Intact genomes are generally too large to sequence. To get around this problem a genome must be cut into lots of small manageable pieces. This semester, two independent study students have isolated the chloroplast genome, mechanically sheared the DNA into small pieces and then placed them into plasmid cloning vectors for sequencing. These plasmids are then grown inside bacteria that will produce the millions of copies that are required for manipulation and sequencing. Today you will extract a plasmid from a bacterial culture. Next week we will use this plasmid to set up a sequencing reaction. Plasmid DNA Extraction Plasmid Extraction Goal – extract the plasmid DNA from a liquid bacterial culture. Materials Resuspension Buffer (P1) Lysis Buffer (P2) Neutralization Buffer (N3) Wash Buffer (PE) Spin Column Elution Buffer (EB) Collection tubes  Introduction Today you will use a commercial kit (Qiagen’s Miniprep) to extract and purify plasmid DNA from your bacterial culture. Plasmids are small circular pieces of DNA that are maintained and replicated separately from the bacterial cell’s genome. Procedure Pipette or pour 2.0ml of culture into a colored microfuge tube. Pellet cells for 1minute at full speed. Pour off the supernatant (growth medium) and blot the open tube against a paper towel. Resuspend your pellet in 250μl of Resuspension Sol