Answered most of the questions. Can you tell me if my answers are correct? Last question of the month. Thank you. I appreciate it.
4. You perform electrophoresis on the product of a PCR and see that instead of the 300-base band you expected that there are five different bands.
What controls the specific sequence to which a PCR primer binds? Complementary sequence and annealing temperature control the specific sequence to which a PCR primer binds.
What are two factors that contribute to the annealing temperature of the primer? Two factors that contribute to the annealing temperature of the primer is primer length and the Guanine and Cytosine content.
What part of your thermal cycle protocol could you change, and how would you change it, to try and get rid of the extra bands?
To get rid of the extra band, we have to increase the annealing temperature.
If, after performing electrophoresis, there were no bands on the gel, what part of your thermal cycle protocol would you change, and how would you change it?
After performing electrophoresis, if there were no bands on the gel then we have to decrease the annealing temperature of the primer
5. If you are using a DNA sequence from a related species to design primers for your species of interest, why is it better to design your primers to complement an exon?
In related species there are similarities but their genetic makeup will not be the same. It is better to design your primers to complement an exon because ??
6. Why is it better to use a 25-base primer than a 10-base primer? Why do you have to have a lower annealing temperature for a 10-base primer?
It is better to use a 25-base primer than a 10-base primer because a 10-base primer is not long enough and may pair with a wrong portion of DNA, which would give wrong results. You have a lower annealing temperature for a 10-base primer because annealing temperature increases when the number of base pairs increase. If the temperature is too high the primer might not bind.
Identify the three steps in PCR and tell the function of each step.
Denaturation - when the double-stranded DNA is heated to 92-95â for about one minute, to separate it into two single strands.
Annealing - temperature of the reaction is lowered to a temperature that is between 45â and 65â to allow the DNA primers to attach to the template DNA.
Extension - temperature of the reaction is raised to a temperature between 45â and 65â and the new strand of DNA is made by adding nucleotides to the ends of the primers.
