You wish to make a recombinant DNA molecule that will contain one piece of pKABO

Question

You wish to make a recombinant DNA molecule that will contain one piece of pKABOO vector DNA and one piece of frog DNA so that you can clone a segment of frog DNA. You want cells containing your recombinant plasmid to be ampr and tets and you want to use enzymes that cut within the insertional marker gene. (Note that tets means that there is no functional tetr gene in the plasmid.) Be sure that your plasmid has the ability to replicate autonomously in a bacterial cell. You do not have to include the entire frog DNA given below in your recombinant plasmid.

1. Which restriction enzymes would be used to clone a segment of frog DNA?

Pick a pair (RsaI and SpeI, RsaI and AluI, RsaI and EcoRI, SpeI and AluI, SpeI and EcoRI, AluI and EcoRI)

2. What is the size of the recombinant plasmid in basepairs?

3. The recombinant plasmid when transformed into E. coli confers resistance to which of the following antibiotics:

Pick one

ampicillin only

tetracycline only

beta-galactocillin only

ampicillin and tetracycline

ampicillin and beta-galactocillin

tetracycline and beta-galactocillin

4. The recombinant plasmid map has many restriction sites. Determine the base pair position of each of the following sites in the recombinant plasmid map:

Fill in recombinant positions

Restriction Enzyme Vector Position Recombinant Position XhoI 240 Answer DraI 935 Answer XmaIII 2780 Answer SacI 3150 Answer RsaI XmaIII SpeI ClaI AluI EcoRI 240 190 185 320 230 XhoI 240 3150 SacI Frog DNA 585 615 RsaI AluI HindIII 640 SpeI EcoRI 2780 XmaIII pKABOC 3878bp 665 680 2175 RsaI DraI 935 1845 EcoRI ori

Explanation / Answer

1. RsaI and EcoRI

2. The size of the recombinant plasmid = 925+3878 = 4803 bp

3. The vector contains Ampr marker. So, the transformed cells will exhibit Ampicillin resistance.

4.

Restriction Enzyme Vector Position Recombinant Position

XhoI 240 240

DraI 935 935+925 = 1860

XmaIII 2780 2780+925 = 3705

SacI 3150 3150+925 = 4075

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