Discussion Questions

·       What are the Gibson overlap sequences in our DNA assembly design? How is an overlap created between the cut pUC19 and the PCR amplified parts of the amilCP gene? See assignment lecture slides

o   The regions with the orange and blue tags are the Gibson overlap sequences.

o   pUC19_PvuII_LrgEnd1: 5’-CCTCTTCGCTATTACGCCAG-3’

o   pUC19_PvuII_LrgEnd2: 3’-CTGCATTAATGAATCGGCCAAC-5’

o   The overlap was created by adding "tails", on the primers flanking the desired region of the amilCP which perfectly matched two regions in the pUC19 vector, which determined where the amilCP gene would be inserted.

·       What is the melting temperature for each overlap sequence and how do they compare to the incubation temperature for the Gibson assembly reaction? See http://biotools.nubic.northwestern.edu/OligoCalc.html

o   pUC19_PvuII_LrgEnd1: 53.8 °C

o   pUC19_PvuII_LrgEnd2: 53.0 °C

o   The melting temperatures are close to and slightly higher than the incubation temperature for Gibson assembly, which is 50.0 °C

·       What is the purpose of each enzyme in the Gibson assembly mix?

o   T5 Exonuclease, a 5’-3’ exonuclease, removes nucleotides from the 5’ end of a double-stranded DNA to create a single-strand 3’ overhang.

o   Phusion DNA Polymerase adds nucleotides to fill in gaps in annealed DNA fragments.

o   Taq DNA Ligase covalently links the annealed DNA fragments and removes nicks.