Gel Electrophoresis lab write-up Title: Restriction Digestion and Analysis of Lamba DNA Objective: 1. To learn how to cut lamba DNA into a series of fragments using restriction enzymes. 2. To learn to separate and sort a large group of DNA molecules according to size 3. Know how to determine the size of each molecule separated by gel electrophoresis. Materials: • Ruler • Marking pencil • DNA from bacteriophage (lambda) • Three restriction enzymes (PstI, EcoRI, and HindIII.) • Micro test tubes • Restriction buffer • Ice • Water (room temp and ice temp) • Styrofoam holder • 5µl pipette • 1µl pipette • Disposable tips • Mini centrifuge • Agarose gel and holder • Loading buffer containing the two dyes (used in gel electrophoresis) • Parafilm • Plastic wash bins Lesson One: Introduction to Restriction Analysis 1. Describe any pattern you might see in the upper sequence of bases: Answer: There is no specific type of pattern associated with the upper sequence of bases. 2. Compare the bases in the upper DNA strand to those in the lower strand. Can you discover any relationship between the upper and lower strands? Describe it. Answer: A always pairs with T; G always pairs with C. 3. Do you notice any grouping of bases that when read towards the right on the upper strand and read toward the left on the bottom strand are exactly the same? Answer: CTTAAG 4. How many base pairs are there to the left of the “cut”? Answer: 4 5. How many base pairs are there to the right of the “cut”? Answer: 10 6. Counting the number of base pairs, is the right fragment the same size as the left fragment? Answer: No, it’s larger. 7. How could you describe the size of each fragment in terms of the number of base pairs in the fragment? Answer: Fragment 1 is a 4-base-pair fragment. Fragment 2 is a 10-base-pair fragment. 8. If the GAATTC palindrome is repeated 4 times on the same piece of linear DNA, and the restriction enzyme that recognizes that base sequence is present and digests the DNA, how many DNA fragments will be produced?
Answer: 5 9. If the GAATTC palindrome repeats are randomly found along the DNA strand, then what can you say about the sizes of the fragments that will be produced when the DNA is digested with a restriction enzyme that recognizes that sequence? Answer: Random sized fragments will be produced. Lesson One: Restriction Digestion (Lab Procedure) 1. Obtain micro test tubes that contain each enzyme solution, lambda DNA, and restriction buffer from the common station. Keep all the stock solutions on ice. 2. Label four micro test tubes L , P , E , and H and place them in the foam Micro test tube holder. L = Uncut lambda DNA (yellow tube) P = PstI restriction digest of lambda DNA (violet tube) E = EcoRI restriction digest of lambda DNA (green tube) H = HindIII restriction digest of lambda DNA (orange tube) • Describe the appearance of the DNA in solution.
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