BIOTECHNOLOGY TECHNIQUES (1016184)

TUTORIAL 1:

SPRING 2012

Ren van Wyk

RESTRICTION ENZYME DIGESTION AND RESTRICTION MAPPING

Many restriction enzymes recognize palindrome sequences in DNA.

N = any nucleotide (G, A, T, or C)

REMEMBER:
Restriction enzyme sites are always given in the 5 to 3 direction.
Isoschizomers recognize the same sequence.
Sometimes different restriction enzymes produce the same overhangs.
sticky-ends.

We say they have compatible

QUESTION 1
A. The restriction map for the plasmid pBR607 is shown below.
the proper bands on the gel.

Draw and label the locations of

Indicate positive and negative electrodes.

QUESTION 2
Draw a restriction map for the plasmid pUC223.

Digestions with EcoRI and BamHI result in the

fragments shown below:

EcoRI: 20 kb

BamHI:

11 kb, 6 kb, 3 kb

EcoRI + BamHI:

8 kb, 6 kb 3 kb

QUESTION 3
Map the plasmid from the fragments shown below:
Pst I:
Eco RI:

4.36 kb
4.36 kb

Sma I: 4.36 kb
Eco RI + Sma I: 3.98 kb, 0.38 kb
Eco RI + Pst I:

3.61 kb, 0.75 kb

Pst I + Sma I: 3.23 kb, 1.13 kb

QUESTION 4
The plasmid in Question 3 is digested with EcoRI, PstI and XmaI.

Draw the electrophoresis gel.

Explain your answer.

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QUESTION 5
You want to clone a piece of DNA into a plasmid.
Bgl II in your freezer.

You have the restriction enzymes Eco RI, Bam HI and

The restriction sites of the plasmid and DNA fragment is shown below.

How will

you insert the DNA fragment into the plasmid?

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Explain your answer.

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QUESTION 6
You want to clone a piece of DNA into a plasmid.
Bgl II in your freezer.

You have the restriction enzymes Not I, Bsp 120 I and

The restriction sites of the plasmid and DNA fragment is shown below.

How will

you insert the DNA fragment into the plasmid?

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Explain your answer.

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