ELECTROPHORESIS+LAB+WG

Key Concepts II: Electrophoresis 1. What is the function of a restriction enzyme and what is its normal function in bacteria? The function of a restriction enzyme is to recognize and cut at a specific DNA sequence. A normal function is to cut up certain phages that are foreign to bacteria. 2. What is the restriction enzyme’s “recognition sequence”? It’s a specific sequence of nucleotides at which a restriction enzyme cleaves a DNA molecule. 3. How did the discovery of bacterial restriction enzymes help genetic engineers? It helps them use the enzymes to cut DNA fragments that could be analyzed and used in a variety of procedures. 4. What do you think it means to “digest” DNA with restriction enzymes? I think digest is meaning to “break down” the restriction enzymes. 5. What process is used to separate pieces of digested DNA? It’s called a recognition sequence.

How does Restriction Enzymes Work? 1. What do you think causes restriction enzymes, like all enzymes, to be highly specific with respect to their substrate? I think its because they cut DNA only within very precise recognition sequences. 2. Write the recognition sequence for the three different restriction enzymes? The red line shows where the enzymes will cut the DNA. 3. What is a palindrome? A palindrome is when the recognition sites are symmetrical. 4. Do you know an example of a palindrome? Write it here. An example of a palindrome is a paper cutter. 5. Why are recognition sequences considered palindromes? They are considered that because on one DNA strand the recognition sequence reads in the opposite direction on the complementary strand.

Cutting DNA with Restriction Enzymes 1. What two ingredients do you mix in the tube? You mix the DNA with the restriction enzyme. 2. How long do you need to incubate the ingredients together and at what temperature? The ingredients need to be incubated for 1 hour. 3. Why is 37C a common temperature for genetic engineering experiments? It’s such a common temperature because it allows you to analyze the DNA better 4. How many microliters in one milliliter? There are 1000 microlitres in one milliliter.

Gel Electrophoresis 1. What determines the direction of DNA movement in a gel? The charge of the molecules determine the direction of the DNA. 2. When and why does DNA move towards the positive pole? It moves toward the positive pole because DNA is a negatively charged molecule, and when gel is applied. 3. What determines the rate of DNA movement in a gel? The restriction enzymes determine the rate of the movement. 4. Which size pieces will have the fastest rate and so move the farthest? The smallest fragments move the most quickly.

http://www.dnalc.org/ddnalc/resources/restriction.html DNA restriction 1. If restriction enzymes are molecular scissors, which enzyme is like molecular tape? DNA ligase is like the molecular tape because it can attach or rejoin DNA fragments with complementary ends. 2. What is EcoR1? It is a restriction enzyme that cuts unevenly. 3. What is the recognition sequence of EcoR1? (Both strands) The recognition sequence of EcoR1 is GAATTC. 4. What are sticky ends? Sticky ends are overhangs. 5. What part of the DNA molecule does a restriction enzyme sever? It severs the sugar-phosphate backbones of the DNA strands. 6. Ligase does dehydration synthesis to reconnect what parts of the DNA molecule specifically? It reconnects the DNA sugar-phosphate bonds.

Gel Electrophoresis 1. Separating pieces of DNA with electricity is much quicker and easier than using…. Its much easier than using gravity. 2. What part of gel electrophoresis is sponge-like? The Porous is sponge like. 3. Which size DNA molecules move through the sponge-like matrix faster? Smaller pieces or larger pieces? The smaller pieces move through the sponge-like matrix faster. 4. What is the range of size that a typical gel electrophoresis set-up can separate? It can separate molecules ranging from several hundreds nucleotides in length to over 10,000 nucleotides. 5. Why is the gel submerged in a salt solution? It’s in a salt solution because it can conduct more electricity, than any other substance. 6. Why are the DNA samples, which are usually colorless, loaded into the gel looking blue? It makes it easier to load the samples, and visually track the DNA migration through the gel. 7. Why does DNA travel towards the positive pole (be specific)? It travels to the positive pole because the negatively charged. Since it’s negatively charged it moved toward the positive pole of the electrophoresis chamber. 8. Describe the movement of pieces of DNA through the gel. The DNA molecules move through the gel by “ reptation”- a reptile-like snaking action through the pores of the agarose matrix. Smaller DNA fragments migrate faster and further over a given period of time than do larger fragments. This is how DNA fragments can be separated by size in an agarose gel.