I finished the gel extraction from the ligations of yesterday. So I decided to spare the 1ul required to measure the concentrations of the DNA for the nanodrop. I also measure the samples from the 3-piece ligations. Here are the resulting concentrations:
- Some nomenclature:
- T#pXX: T stands for tetherable; #indicates which adapter is in the final product (either 5’biotin or internal-biotin); pXX is the plasmid name for the adapter (either pRL or pALS)
- T5pRL (~5.4kb)
- 23.8ng/ul
- 6.6nM
- TIpRL (~5.4kb)
- 9.3ng/ul
- 2.6nM
- T5pRL (~5.4kb, 3-piece result)
- 14.7ng/ul
- 4.1nM
- TIpRL (~5.4kb, 3piece result)
- 50.4ng/ul
- 14nM
- T5pALS (~8.4kb)
- 23.6ng/ul
- 4.3nM
Overall a much better yield than I expected, but I don’t completely trust the nanodrop so take these results to be whatever you want. In my experience I’ve never had good tethering efficiency with anything less than about 100pM DNA so I would recommend dilution of each of these about 1:10 for tethering purposes.