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Image Credits: https://www.dana-farber.org/research/departments-centers-and-labs/integrative-research-centers/chemical-biology-program/protein-protein-interactions/
Synthesis
Synthesis
The nanobodies are about 600bp long which is too difficult to synthesize with a single oligo. The yield of full length oligos decays exponentially as the fragment size increases. We thus broke it down into 4 pieces with 20bp adjacent overlaps before assembling them. Below are 3 different assembly methods we used to try to maximize yield and specificity.
Image Credits: https://www.idtdna.com/pages/education/decoded/article/oligo-synthesis-why-idt-leads-the-oligo-industry
Method 1
Method 1
Phosphorylation-Ligation
Method 2
Method 2
Initial Incubation
Mechanism for Method 2
method 3
method 3
Slow Ramping PCR
Best method
Best method
Just by looking at the gels, only method 3 gave a bright, isolated band of the desired size. The other two were neither specific or gave the desired yield.
We were able to quantify the amount of desired product based on the brightness of the sample. A linear regression constructed from the ladders was used to correlate brightness (used ImageJ software) with concentration. From this about 1/3 of the sample using method 3 gave the desired band. This method should be used for scaled up in vitro translation reactions.
Credit: Sophia Han
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