Abstract:
The isolation of large amounts of plasmid DNA from insert-containing clones is necessary because subsequent DNA sequence analysis requires high levels of pure starting DNA. We will be using a moderately expensive plasmid isolation kit (Promega, Inc.) because it produces DNA compatible with our sequencing apparatus. As compared with the rapid mini-prep, large-scale plasmid isolation methods all share three basic parts: (1) a way to gently lyse the cellular hosts; (2) a way to crudely separate plasmid from the total cell extract; and (3) a way to purify and concentrate plasmid DNA.
In order to compare your samples using DNA fingerprinting methods - you will set up restriction digests based on some moderately tricky pre-lab analysis. Recall that restriction enzyme digest reactions require template DNA, enzyme(s), an appropriate enzyme buffer, and some water. Today, you will set up uncut samples, single digests, and double digests using interesting enzymes that should reveal more complex cutting patterns than those produced by EcoRI last time. As you will see, there are many challenges to this. For example, two enzymes we want to cut at the same time may function to lesser extents in non-ideal buffers. Charts are available in the form of company literature (in our case: Gibco BRL Life Technologies) to assist us when faced with the problem of making recipes and figuring these kinds of compatibility issues out. Additionally, you will analyze the enzyme's recognition sequence and calculate - based on probability - how many times each enzyme is predicted to cut the clone. Does the pattern you obtain match this prediction - why or why not?
See full Plasmid Isolation Procedures at
National Science Foundation
Western Oregon University
Yellowstone National Park
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