r-dna technology

Experiment 5

Aim of the Experiment

To perform restriction digestion of Lambda (λ) DNA using EcoRI and HindIII enzymes.

Introduction:

In 1978, the Nobel Prize for Medicine was awarded to Werner Arber, Daniel Nathans and Hamilton Smith for their discovery of restriction endonucleases, which led to the development of recombinant DNA technologies. The first practical use of restriction enzymes in science and medicine was the manipulation of E. coli bacteria to express recombinant human insulin for the treatment of diabetis. The restriction enzymes have been discovered in many different bacteria and other unicellular organisms. These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of bases that they recognize.

Principle

Restriction Digestion involves fragmenting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases commonly known as Restriction Enzymes (RE). Because of this property restriction enzymes are also known as molecular scissors. The restriction enzymes are named from the cellular strain from which they are isolated. Restriction enzymes recognize specific sequences in the double stranded DNA molecule (for example GATATC) and then cut the DNA to produce fragments, called restriction fragments. The target site or sequence which the restriction enzyme recognizes is generally from 4 to 6 base pairs, arranged in a palindromic sequence. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. The size of these fragments is measured in base pairs or kilobase pairs (1000 bases).

Common Restriction Enzymes:

Every restriction enzyme has unique target sites for digestion. Lambda DNA has multiple restriction sites for both EcoRI and HindIII which result into several fragments of varying sizes.

Procedure

Application of Restriction Enzymes:

· Construction of recombinant DNA molecules

· Mapping the locations of restriction sites in DNA

· Southern Blot Hybridization

· Construction of DNA Libraries

Important Instructions:

i. Read the entire procedure carefully before starting the experiment.

ii. The restriction enzymes are temperature sensitive and should always be placed on ice during the experiment.

iii. While performing the experiment place the assay buffers and restriction enzymes on ice.

iv. Use fresh tip while adding different solution to the tube.

v. While preparing the reaction mixture the enzymes should always be added at last.

Procedure:

a. Before starting the experiment, crush ice and place the vials containing Lambda DNA, Restriction Enzymes and Assay Buffers onto it.

b. In this experiment, Lambda DNA is digested with two restriction enzymes; EcoRI and HindIII.

c. Set up the reaction mixture as follows:

After preparing the two reaction tubes, mix the components by gentle pipetting and tapping.

A) Incubate the tubes at 37^oC for 1 hour.

B) After 1 hour incubation, immediately place the vials at room temperature (15-25^oC) for 10 minutes.

C) Run the samples on agarose gel as given below.

Agarose Gel Electrophoresis:

Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use.

Preparation of agarose gel: To prepare 50 ml of 1% agarose gel, measure 0.5 g agarose in a glass beaker or flask and add 50ml 1X TAE buffer. Heat the mixture on a microwave or hot plate or burner by swirling the glass beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60^oC. Add 0.5 μl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

NOTE: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile gloves is recommended.

Loading of the DNA samples: Load 3 μl of ready to use DNA Marker into the well 1. To prepare sample for electrophoresis, add 2 μl of 6X gel loading buffer to 10 μl of DNA samples. Mix well by pipetting and load the samples into the well.

Electrophoresis: Connect the power cord to the electrophoretic power supply according to the conventions: Red-Anode and Black- Cathode. Electrophorese at 100-120 V and 90 mA until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

*Molecular biology grade water is recommended (Product code: ML024).

Observation & Result:

Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator.

Fig 1: λ DNA digested with EcoRI &HindIII

Lane 1: Undigested λ DNA

Lane 2: λ DNA digested with HindIII

Lane 3: λ DNA digested with EcoRI

After running the digested samples on agarose gel, look for the digestion pattern of the two restriction enzymes. Compare the size of each fragment with that of the DNA marker.

Reference Material

https://biologyreader.com/restriction-enzyme.htm

https://vlab.amrita.edu/?sub=3&brch=77&sim=694&cnt=1

Interpretation & Conclusion:

Restriction digestion patterns of lambda DNA obtained upon treatment with EcoRI and HindIII are markedly different which demonstrates the fact that each restriction enzyme recognizes and cleaves only a specific base sequence unique to it. The size of the fragments can be determined by comparing with that of the DNA marker ran on the same gel.

Questions:

Q1. What would be the reason if you observe partial or no digestion of DNA?

Q2 What is STAR Activity?

Q3. What is the reason for insufficient resolution of bands in the gel?