Create an experiment to test what happens | Homework Helpers

You have a 4kb ampicillin-resistant plasmid with one BamHI site and a single HindIII site 1.5kb away from the BamHI site (shortest distance).

you cut one microgram of the circular DNA (which we will assume has no nicks in it) with BamHI, and one microgram with HindIII. You heat-inactivate the enzymes (if you prefer you could purify the DNAs by phenol extraction and ethanol precipitation), mix the two linear DNAs together, denature (by boiling for 2 minutes) and then cool slowly to allow hybridization (these conditions should allow only hybrids with perfect or near-perfect base-pairing). What products would you expect to find?

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If you took a small amount of the product and transformed competent cells, which, if any, of the DNA species would you expect to give rise to transformed colonies? Explain.

Imagine that the plasmid described above has a single EcoRI site (say 1.0kb from the BamHI site and 0.5kb from the HindIII site) and that you had an identical plasmid except for a 1bp change (small enough that it will not affect hybridization properties) that eliminates the EcoRI site (GAGTTC instead of GAATTC).

Design an experiment to test what happens (i.e. what is the final plasmid DNA content of cells in a transformed colony) if a competent cell is transformed with circular double-stranded plasmid DNA where there is a mis-match (e.g. a G-T base-pair instead of an A-T base-pair). Be sure to explain what you do, what you measure and how you interpret the results.

What result do you expect? Explain why you expect that result?

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