Explore BrainMass

ampicillin-resistant plasmid

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 tofind?

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?

Solution Preview

When you cut one microgram of circular DNA with BamHI you would get a 4kb linear fragment. Similarly, when you cut with HindIII, you would again get a 4kb linear fragment. Upon denaturing the DNA, the double stranded linear fragments will be single stranded; therefore, there will be 4kb linear fragments of BamHI upper and lower fragments and similarly upper and lower fragments of HindIII. Upon hybridization, you would get the upper and lower fragments of BamHI hybridized with a 3996bp double stranded and the remaining 4bp overhangs on either ends of the linear double stranded DNA (total 4kb size). Similarly, the HindIII fragments will hybridize and form a double stranded DNA with a total DNA of 4kb in size. But since, there will be homology ...

Solution Summary

This job examines an ampicillin-resistant plasmid experiment.