1. Design primers that will amplify the following region of DNA. The primers should be 15 bases in length and indicate the 5’ and 3’ ends of the primers.

5’-GCGATCGGCTCAACTCGATCGAGATCCTCCTGATGATGCGCAGCCGCGTTAA-3’

2. In PCR we employ a three-step thermal cycle. What is the purpose of each step and what approximate temperatures are used?

3. Write the ligated “Gibson” assembly product of the DNA fragments below.

5’-ATGCGTACGTACGTC-3’   5’-ACGTCGTTCCGACGCG-3’

3’-TACGCATGCATGCAG-5’   3’-TGCAGCAAGGCTGCGC-5’

4. What is the key to enabling the template plasmid to be degraded following site-directed mutagenesis?

5. In “Golden-gate assembly” we use sticky ends to assemble multiple inserts into plasmids. How do we ensure each pairing of sticky ends is specific?

6. In “CRISPR” how is Cas9 targeted to specific DNA sequences for cutting?