RGT Assignment 2

RGT Assignment 2

1. You have grown up a liter of cells containing a plasmid and isolated the plasmid using the alkaline hydrolysis method. When you run a sample on an agarose gel you find your plasmid bands as well as a large amount of RNA at the bottom of the gel. Describe three methods that could be used to recover the DNA without the RNA.

2. You have just isolated several DNA samples and want to quantify the DNA. You take a scanning spectrum and determine the absorbance at 260, 280, and 320 nm. The table below shows the absorbance results.

Sample	A260	A280	A320
1	0.222	0.111	0.001
2	0.413	0.301	0.001
3	0.500	0.363	0.200
4	0.824	0.412	0.002

a. Which samples are pure nucleic acid?

If any have contaminants, how would you purify them?

b. Accurately determine the concentration of the nucleic acids in all the samples possible. If the determination of the DNA concentration requires that something be done to the sample before quantification, note what that procedure would be.

c. Which samples could be contaminated with RNA?

How did you determine that?

3. A fragment of foreign DNA (that had been cut with restriction enzyme C) is inserted into the pAlpha vector below at restriction enzyme site C. Determine the restriction map of this insert from the restriction digest fragment size data listed below.

Fragment Sizes (bp) of Recombinant pAlpha cut with restriction enzymes

A	B	C	D	E	F	AB	DB	DA	DC	EA	EB
250 4500	750 1000 3250	750 1250 3000	750 4250	5000	500 4500	250 500 750 3250	250 500 1000 3000	250 750 3000	750 500 3000	250 1500 3000	250 750 1000 3000