From a chemical point of view, proteins are by far the most structurally complex and functionally sophisticated molecules known.--- Molecular Biology of the Cell, 5th
Joseph Jacobsonfrom MIT gave us a overall introduction on DNA synthesis and its evolution. Then, the founder of Twist Bioscience Emily LeProust talked about a little history of DNA synthesis, the technology Twsit use and their new business plan.
In this part, we will prepare and order the primers that will generate a library of mutated amilCP expressing E. coli cells. We will use Gibson Assembly to insert our mutated gene into a plasmid, which in turn will be transformed into electrocompetent E. coli cells. First, let's understand what is Gibson Assembly and how to design primers for it.
1. Primer design to linearize plasmid backbone
About pUC19: pUC19 is a prasmid of vector.This prasmid vector is used for DNA sequencing by dideoxy-method.
Use NuPack to help select 18 bp priming sites that amplify a ~2.25 kb region of pUC19 (NEB) immediately upstream of the plac promoter and downstream of the start of lacZalpha. The resulting amplicon excludes the plac promoter and n-terminus of lacZalpha, which enables you to swap in a gene of interest under the control of a promoter of interest using Gibson Assembly later on in your workflow. Design one pair of oligos that prime optimally and another that prime poorly. Describe the PCR thermocycling program that uses Phusion polymerase (NEB) for these pairs. Crucially, determine annealing temperatures and extension times.
Good pUC19 Primers
Primer-1 GTTAAGCCAGCCCCGACA
Primer-2 GAAACCTGTCGTGCCAGC
BAD pUC19 Primers
Sucky-Primer-3 ACAGCTTGTCTGTAAGCG