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.

Restriction enzymes AnswerBamHI and SpeIBamHI and HindIIIBamHI and PstISpeI and HindIIISpeI and PstIHindIII and PstI would be used to clone a segment of frog DNA.

The size of the recombinant plasmid is Answer bp.

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

ampicillin only

tetracycline only

beta-galactocillin only

ampicillin and tetracycline

ampicillin and beta-galactocillin

tetracycline and beta-galactocillin

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

Please answer all parts of the question.

Restriction Enzyme Vector Position Recombinant Position NotI 75 Answer AluI 1050 Answer BamHI 1525 Answer PstI 2150 Answer 2150 PstI 1900 KpnI 1525 BamHI 1200 EcoRI NotI 75 pKABOO 2500bp or BamHI KpnI Spel EcoRI Hindi II PstI 350 T 400 T 200 300 700 Frog DNA BamHI 550 600 Spel 750 HindIII 900 PstI 915 ClaI AluI 1050

Explanation / Answer

Restriction enzymes to be used would be SpeI and HindIII. Because, restriction sites chosen should be unique i.e. it appears only once in the plasmid and target DNA (here frog DNA) so that the restriction enzymes cut the desired region and do not act on other regions of the plasmid or target DNA. SpeI and HindIII occurs once in the plasmid as well as target DNA whereas there are multiple sites for BamHI and PstI on the plasmid.

The size of the recombinant plasmid would be 2500 (original plasmid size) - 150 (DNA region between SpeI and HindIII that will be removed) + 500 (frog DNA region that would be inserted) = 2850 bp

The recombinant plasmid would be ampicillin resistant. The insertion of frog DNA in the tetracycline resistance gene would interrupt the gene and thereby do not produce a functional gene for tetracycline resistance. Beta-galactocillin gene isn't present on the plasmid.

Restriction enzyme - Recombinant position

NotI - 75

AluI - 1400

BamHI - 1875

PstI - 2500

Calculation: The isertion of 500bp frog DNA has added 500 bp and removed 150 bp from original plasmid. hence, there is a difference of 350 bp in the original vector position and recombinant position which needs to be added in the enzyme restriction sites which occurs after the first modification site i.e. SpeI site. NotI occurs in the same position whereas rest of the enzymes will occur 350 bp away from original position.

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