Question 1: As you were setting up your PCR reaction to amplify a specific fragment of DNA, you became distracted by your lab mates (Dr. Bunsen and Beaker), who accidently lit Beeker’s hair on fire. You added all the ingredients for PCR (Template DNA, Taq polymerase, dNTPs, a buffer) except you only added one primer instead of both. You then set up the reaction to cycle 30 times.
a. Will this reaction produce a billion copies of double stranded DNA? Explain.
b. Assuming that you started with only one copy of template DNA in your PCR reaction, how many copies of the target loci on each strand will you make after the reaction is finished?
Question 2 (4 pts): In a typical sanger sequencing reaction, the ddNTPs are added at a low concentration. What would the gel look like if you added 100X more ddNTPs. Please
draw
what you would see, and explain it in words.
Question 3 (7 pts): The restriction enzymes AgeI and AvaI recognition sites are shown below. (NOTE, the arrows represent where the enzymes actually cut the DNA for each strand).
AgeI: 5’-ACCGGT-3’ AvaI: 5’-CCCGGG-3’
3’-TGGCCA-5’ 3’-GGGCCC-5’
Sequence 1: 5’-GGACCGGTCC-3’ Sequence 2: 5’-AACCCGGGTT-3’
3’-CCTGGCCAGG-3’ 3’-TTGGGCCCAA-5’
A. Write out the DNA fragments that are formed after cutting the above DNA sequences with:
Sequence I with AgeI (2 pts):
Sequence 2 with AvaI (2 pts):
B. Based on your DNA fragments from parts A and B, if you put all those fragments together into the same tube, and added DNA ligase, could it be possible to make an entirely new DNA fragment using the sticky ends? If so, draw out all the new fragments formed. If not, explain why not?