Digestion of bacteriophage lambda DNA using a restriction enzyme

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Digestion of bacteriophage lambda DNA using a restriction enzyme.

1. Objective :

Digestion of bacteriophage lambda DNA using a restriction enzyme.

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2. Introduction :

Restriction enzymes have been described in Chapter 3 of this textbook. They are endonucleases which recognize and cleave the specific DNA sequences called restriction sites for example, £coRI (isolated from Escherichia coli) that recognizes and cleaves the sequence 5′-GAATTC-3′ to generate cohesive or sticky ends.

Similarly, Hind III isolated from Haemophilus influenzae recognizes and cleaves the sequences 5′-AAGCTT-3′ to generate cohesive or sticky ends.

Enzyme activity is represented as IU (International Unit). One unit of a restriction enzyme is the amount of enzyme required to completely digest one microgram of lambda DNA [in a reaction volume of 50 pi in one hour under optimal conditions of salt, pH and temperature (about 37°C for most restriction enzymes)].

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3. Principle :

Phage lambda (λ) DNA is a liner double-stranded DNA molecule containing 48,502 base pairs (bp). Its DNA becomes circularized after release (inside the cell of E. coli) at a cohesive site called COS site. It contains five recognition sites for EcoRI and seven recognition sites for HindIII.

The complete digestion of lambda DNA with EcoRI results in 21, 226, 7421, 5804, 4878 and 3530 bp long six DNA fragments. Similarly, a complete digestion of lambda DNA with Hindlll results in eight DNA fragments viz., 23, 130, 9416, 6557, 4361, 2322, 2027, 564, 125 bp long fragments.

4. Requirements :

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i. Lambda DNA, restriction enzyme such as ZscoRI or HindIII

ii. Assay buffer for restriction enzyme, sterilized water, Tips, Eppendorf tubes, micropipettes

iii. Agarose gel electrophoresis apparatus

5. Procedure

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(i) Always keep restriction enzyme (EcoRI or HindIII), substrate (X DNA) and assay buffer in an ice bucket.

(ii) Take 2-5 pg of the lambda DNA as substrate in an eppendorf tube and dissolved in an appropriate volume of water.

(iii) Add 2 pi of about 10X assay buffer (available with the restriction enzyme) to the DNA in the eppendorf tube, followed by respective enzyme (5-12 units of EcoRI or 10-25 units of HindIII depending upon the amount of DNA used in the reaction step (ii).

(iv) Add sterilised water to make the final volume of reaction mixture to 20 pi. Centrifuge gently or mix by tapping with fingers.

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(v) Incubate the reaction mixture for 1 hour at 37°C in a water bath or incubator.

(vi) As described in the plasmid isolation experiment, in the mean time prepare 1% agarose gel for loading and electrophoresis.

6. Results:

A single band is seen in the lane in which sample ‘B’ (A, DNA) has been loaded. While the second lane in which sample ‘A’ has been loaded shows multiple bands. This reveals the cleavage of sample ‘B’ by the respective restriction enzymes.

The exact number and size of the bands obtained depend on the restriction enzymes used for digesting the lambda DNA (sample ‘B’). As explained earlier, 6 DNA fragments are observed if £coRI is used and 8 DNA fragments are observed after using Hindi I).

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