Cut, Copy, Measure

A definite sequence, actually searched

The plasmid below is a definite sequence of 2,738 bases, and the cut sites were not looked up — they were found by searching that sequence for each enzyme's recognition site. Pick an enzyme and see where its site occurs, including the case where it occurs nowhere at all.

The fragments, and the check

Reading a gel properly

Every textbook says distance depends on the logarithm of the size. Here is what happens when you take that seriously, and what happens when you do not.

Three unknown bands, sized both ways

Doubling is not the whole story

The DNA doubles every cycle from the very first one. The fragment you actually want does not exist at all for the first two cycles. Step through and watch the two quantities separate, then converge.

Putting a gene into a cell

Learning check

Answer these in your own words before you open the answers.

1. Explain what a restriction enzyme does, and why its recognition sequence reads the same on both strands.
2. A circular plasmid is cut at 4 sites. State how many fragments result, and how many there would be if the plasmid had been cut open into a line first.
3. State the check you should always perform on a set of fragment lengths, and explain what it tells you.
4. Explain how gel electrophoresis separates DNA fragments, and state which end of the gel the DNA moves toward.
5. Explain why a calibration curve for a gel is plotted against the logarithm of the size, using a figure from the second tab.
6. A gel shows 3 bands. Explain why you cannot conclude the digest produced exactly 3 fragments.
7. Explain what determines which region of DNA is amplified by PCR.
8. “PCR doubles the amount of your product every cycle from the start.” Evaluate that statement.
9. Bacterial transformation succeeds in about 1 cell in 10,000. Explain why that is not a problem in practice.