11. What are the 3 steps in the PCR thermocycle? What happens in each step? Plea
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11. What are the 3 steps in the PCR thermocycle? What happens in each step? Please indicate the approximate temperature (in Celsius) for each step. 12. What are the two main functions that primers serve? 13. DNA migrates to which electrode? DNA mto anod e 14. What is the purpose of ethidium bromide (EtBr)? How does it work? 15. What is the purpose of the TAE buffer and what does TAE stand for? 16. What are the purposes of glycerol and Bromophenol Blue dye? 17, How would you make 200 mL of 1.5% agarose in 1x TAE buffer? 18. If you have a 40X TAE stock and need to make 500mL of Ix TAE, how much 40X TAE and how much dH20 would you add?Explanation / Answer
Answer 11. The 3 steps of PCR thermocycle are as follows.
1. Denaturation: In this step temperature is raised to a point where the hydrogen bonds keeping the DNA intact is broken. The temperature for this step is 94-98°c for 20-30 seconds.
2. Annealing: In this step temperature is decreased to allow the annealing of primers to each of the single stranded DNA strands. The temperature for this step is 50-65°c for 20-40 seconds.
3. Extension: Temperature in this step depends on the polymerase used. Most commonly used polymerase is taq polymerase and it requires a temperature of 72-75°c. In this step DNA polymerase synthesizes the DNA strand complimentary to the DNA template strand by adding free dNTPs in the 5' to 3' direction thus elongating the DNA strand and multiplying the DNA in the mixture. There is one final step where reaction cools down to 4-15°c before ending the cycle.
Answer 14. Ethidium bromide is a DNA intercalating agent used as a fluorescent tag for nucleic acids in labs. It is used to visualize the DNA in gel. When exposed to UV rays it fluoresce an orange color. It is a potent mutagen and should be used carefully.
Answer 15. TAE buffer is a mixture of tris base, acetic acid and EDTA. It is used in agarose electrophoresis to seperate nuclic acids. TAE buffer is made up of tris acetate buffer having a pH of 8.3.
Answer 16. Glycerol and bromophenol blue dye is used in the loading dye mixture for agarose gel electrophoresis. The dense property of glycerol renders the sample heavier than the running buffer thus allowing it to load properly and the blue colour of bromophenol blue dye lets the user visualize how fast is the gel running.
Answer 17. To make 200 mL of 1.5% agarose in TAE buffer we need to add 3 grams of agarose powder to 200mL of 1X TAE buffer.
Answer 18. We need 487.5 mL of dH2O and 12.5 mL of 40X TAE to make 500 mL of 1X TAE.
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