AFM test sample: DNA origami rectangles

Testing the achievable resolution of atomic force microscopes under realistic conditions is important in order to know the possibilities but also the limits of real applications. With GATTA-AFM nanorulers, now adequate test samples are finally available. These DNA-based samples are in a well-defined rectangular shape with a size of 70 nm times 90 nm and a height of 2 nm. The GATTA-AFM nanorulers represent accurate and highly parallelized structures and are therefore perfectly suited to optimize and test the resolution of atomic force microscopes.

The amount of material is sufficient for at least 10 AFM surfaces, densely packed with DNA origami rectangles. The ladder like substructure in the middle of the GATTA-AFM sample shows a line spacing of around 6 nm. Samples are shipped in solution.

Immobilization of GATTA-AFM Nanorulers

Immobilization strategy using mica sheets and imaging under liquid conditions
I) Use a freshly cleaved mica sheet (for instance, a 12 mm mica disc* glued to a metal puck) and incubate it with 20 μl of folding buffer (1x FB: 1x TAE supplemented with 10 mM MgCl2) for 1 minute. The mica sheet must be clean and flat without defects or cracks. Dispense the buffer homogene-ously to the whole mica surface.
ll) Add about 10-50 fmol of GATTA-AFM sample (for instance, this equals to 1-5 μl volume for a 10 nM sample concentration) to the buffer on the mica. Tilt the mica sheet carefully in all directions to dis-tribute the sample all over the surface and let it incubate for another 2 minutes.
lll) Your GATTA-AFM sample is ready to use. More buffer (1x FB) can be added if required by your ima-ging system. Magnesium ions might be replaced by other strong bivalent ions (monovalent ions like sodium should be avoided). Please be aware that changing the ions might require special safety measures and can influence shape and stability of the DNA nanostructure.
*Other dimensions and shapes for mica sheets can also be used. Please adjust the used volumes and amounts accordingly.

Imaging under dry conditions
lV) Prepare two watch glasses or flat bowls, one with 70/30% ethanol/water, the other one with pure ethanol.
V) Prepare your sample according steps i)-iii).
Vl) Take a tweezer and dip your mica sheet from step iii) briefly into the ethanol/water bath. Directly afterwards transfer it to the ethanol bath and keep it there for a few seconds.
Vll) Take the mica sheet out of the ethanol bath and let it dry (air dry or under gentle N2 stream). If the sheet is dry it is ready to use.