Texas A&M University-Corpus Christi CHEM4402 Biochemistry II Laboratory Laboratory 7: Plasmid DNA Isolation and Restriction Enzyme digestion Note* - Please bring your laptop and an electronic copy of the formatted GFP sequence you obtained for GFP from our second lab, database searching, Over the past few weeks we have been constructing a recombinant DNA molecule from a plasmid DNA and our GFP PCR amplification product. Today we will build on last week’s transformation experiment and actually isolate the GFP-containing plasmid DNA from its E.coli host. To do this, a single positive (white) colony is selected from the LB-agar-ampicillin plate and placed in a nutrient media (Luria Broth, or simply “LB”) with an antibiotic (ampicillin) to prevent growth of any non-transformed bacterial cells that find their way into the solution. The inoculated media is then allowed to grow overnight while shaking at 37 o C. Shaking helps to provide oxygen to the cells and prevents them from clumping together and settling. 37 o C is the optimal temperature for cell growth. A single overnight incubation is sufficient for an inoculation of E.coli cells to increase in number a billion-fold. This quantity of cells will provide milligram quantities of DNA, enough for experiments using PCR, restriction enzymes, sequencing or other techniques. To complete the isolation of plasmid DNA from overnight cultures in a single day, your instructor has already selected a representative positive (white) clone and performed an overnight incubation to allow enough cells to grow. All groups will be isolating plasmid DNA from this culture. To analyze the isolated plasmid DNA, we will perform a restriction enzyme digest of the DNA, followed next week by electrophoresis on agarose gels for analysis. Restriction enzymes are DNA endonucleases; they cut DNA molecules at specific sequence target sites. By comparing the fragments generated from an enzyme digest to those expected from known nucleotide sequence data, a research scientist can verify whether their plasmid DNA preparation does indeed harbor the cloned DNA product of interest. To determine what fragments should be produced from our
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