Restriction digestion of DNA PRACTICAL kit guide pdf
Restriction digestion of DNA PRACTICAL kit guide pdf
Restriction Analysis —
Links to Biotechnology The techniques introduced in this exercise form the basis of recombinant DNA technology techniques,
Eukaryotic and prokaryotic gene structure
DNA fingerprinting, and forensic DNA analysis. This kit introduces students to some important principles of genetic engineering. Specifically, the functions of restriction enzymes and their use as molecular biology tools will be stressed. Using agarose gel electrophoresis, students will examine the digestion patterns, analyze the migration distances, and determine the sizes of unknown DNA fragments. Restriction enzymes were a catalyst for the molecular biology revolution,
Lesson 1 Introduction to Restriction Analysis Consideration 1 Pour gels and digest DNA samples
Lesson 2 Agarose Electrophoresis Considerations 2 and 3 Load and run gels Stain gels (record results and dry gels if using the quick staining protocol)
Lesson 3 Analysis of Results Record results and dry gels (if using the overnight staining protocol) Analyze results Complete analysis questions Generate standard curve Discuss results
Eukaryotic Gene Structure
Lesson 1 Restriction Digestion
1. Obtain micro test tubes that contain each enzyme stock solution, lambda DNA, and restriction buffer. Keep all the stock solutions on ice.
2. Obtain one of each colored micro test tubes and label them as follows: yellow tube: L (lambda DNA) violet tube: P ( Pst I lambda digest) green tube: E ( Eco RI lambda digest) orange tube: H ( Hind III lambda digest)
3. Using a fresh tip for each sample, pipet the reagents into each tube according to the table below:
tubeDNA buffer
Pst I
Eco RI
Hin dIII
L 4 µl 6 µl – – – P 4 µl 5 µl 1 µl – – E 4 µl 5 µl – 1 µl– H 4 µl 5 µl – – 1 µl
4. Mix the components by gently flicking the tube with your finger and tapping gently on the table to collect liquid to the tube bottom. Pulse-spin the tubes in a centrifuge to collect all the liquid to the bottom, or tap them gently on the benchtop.
5. Place the tubes in the floating rack and incubate for 30 minutes at 37°C or overnight at room temperature.
6. After the incubation, place the samples in the refrigerator until the next laboratory period, or proceed directly to step 2 of Lesson 2
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