To self-assemble the DNA origami structure in one pot begin by preparing a mixture of 2.5 nanomolar of the circular scaffold strand M13mp18 and 100 nanomolar of the 201 short oligonucleotide staples in TAE buffer supplemented with 15 millimolar magnesium chloride. Then adjust the total volume of the solution to 100 microliters using ultrapure water. Anneal the solution in a thermocycler using a temperature gradient.
Start by rapidly heating to 80 degrees Celsius and follow the displayed temperature gradient program step by step. To purify the mixture from excess staples add 100 microliters of the DNA origami solution to a 100 kilodalton molecular weight cutoff amicon filter. Followed by adding 400 microliters of ultrapure water.
Then centrifuge at 6, 000G for eight minutes at room temperature. After centrifuging, remove the filter and flip the tube to discard the filtrate. Add 400 microliters of ultrapure water to the filter and centrifuge as demonstrated previously.
To collect the purified nanostructure solution. Flip the filter upside down in a new tube and centrifuge at 1, 000G for two minutes at room temperature following the manufacturer's instructions. Proper assembly of the DNA origami fork was ensured using atomic force microscopy or AFM imaging.
As expected, most forks were solid with no broken arms. However, the bridge between the arms was difficult to image because of its small diameter and high flexibility.
