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80 nt inserts with P-free nicks

Synthetic DNA 40 nt long is only ~$ 6.
-Longer oligos are significantly more expensive.
-Double stranded DNA means 2x the cost.
-Synthetic DNA means any sequence is possible.
Inserts 40 nt long gives you peptides 7-8 AA long.
-Need to account for upstream/downstream restriction sites.
-The restriction sites make 16 nt unavailable for coding.
-Not much peptide design flexibility with only 7-8 amino acids.
With 80 nt, now peptides can be 20 AA long, longer than a GFP barrel stave.
-Normally, two 40-mers would have to be ligated.
-Requires a separate kinase step to install a 5' phosphate.
-We take advantage of the fact that complementary strands have built in stability.
The diagram below shows how 5 oligos can be assembled to give 80 nt inserts.
-Does not require a kinase step, E. coli can be transformed with plasmids having P-free nicks.
-Modular approach: reuse unchanged oligos, order new oligos with changes only.
-A brand new 80mer insert costs $20-$30, with mutations to it costing as little as $12.


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FA diagram