1. PCR
a. (7) Primer Design: Take the DNA sequence below, and choose a 150 bp sequence to amplify, starting at nucleotide 65. Define that sequence in brackets. Define the Forward & Reverse Primers (20 bp each), and write the sequence for each primer.
1 ggatcctggaaccagagttacagaaaggtgtgagctgctgtgtgggtgctaggaatcaaa
61 cctgggtcttttagaggagcagccagtgcatttagctgctgagccatctctcgggccctt
121 agttcaagtgttttaaatgatgcattttatgctttgtatacgtaatgaaataaaatgatt
181 tttttcccagaaaagattaaggagcccactaaagactatgtaactagaccgtggccaagc
241 cagatttgaacagaacctgcaatgtctcttgaatggaaactatgcatacctgagcagcca
301 ccaaaatttaggacatccggttcttggcatccctgtcttgtgtgaccttacacagactga
361 cctttgaagatctcgttctcttcagctattaaataataagacttaagtatctttgacggg
Forward (5’ – 3’):
Reverse (5’ – 3’):
b. (2) What would happen if you forgot to put in the MgCl2 into the amplification reaction?
2. Real-Time PCR
a. (4) Based on the amplification plot:
i. What conclusions can you make regarding Sample A?
ii. Which sample has a higher starting DNA concentration, Sample B or C?
a. (4) Based on the data:
iii. Estimate the concentrations of samples B & C:
Sample Ct Sample Ct
100 ng/ulstd 15 Sample A 19
50 ng/ulstd 16 Sample B 16.5
25 ng/ulstd 17 Sample C 15.25
12.5 ng/ulstd 18
6.25 ng/ulstd 19
3. Discuss the below standard curve.
a. (3) What is wrong with this curve? What could you do to still use this plate data?
a. (2) What is one hallmark of a “good” standard curve, and why is it important to have a good standard curve?
4. (2) Draw a line where you think the Cycle Threshold (Ct) should be, and justify your answer.
5. (3) If you start with 100 pg of genomic DNA, how many copies of your target fragment will you have theoretically at the end of 30 PCR cycles? Assume diploid chromosomal number, 3.7 pg of DNA per cell and 100% efficiency of doubling for all cycles.
Steps:
1. How many copies of a diploid target are theoretically in 100 pg of genomic DNA?
2. Calculate the logarithmic growth of X copies (defined in step 1) over 30 exponential cycles.
6. (3) Make up a 20 nt primer that has a hairpin loop. Show both linear sequence and structure.
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