OhioMOd2013:Methods/origamifolding

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Folding Reaction

A folding reaction is a mixture containing scaffold DNA, staple DNA, water, a buffer mastermix, and magnesium ions. The folding involves subjecting the sample mixture to thermal denaturation followed by renaturing. We will usually have the folding reactions occur in PCR strip tubes of either 50-100 μL aliquots.

Pre-stocks

The pre-stock is a pool of oligonucleotides that contain specific components of the origami structure. They are therefore sub-sets to the intended structure. We divide the staples into specific pre-stocks so that in the event we change a specific component to the origami structure, then only that single working stock will need to be changed. Here is an example pipetting guide that is used to prepare the pre-stocks. This guide includes from what plate and range of wells the staples were taken from, the name of the pre-stock, the number of oligos, their associated concentration, and what element of the structure the pre-stock is part of.

index plate from well to well name no of oligos conc.(μM) element
p01 p085 A1 H12 CoreP085 96 1.04 core 1
p02 p086 A1 D5 CoreP086 41 2.44 core 2
p03 p086 E1 F1 Neighbors 13 7.69 Neighbors
p04 p086 G1 H1 Polymers 13 7.69 Replaced by polymers

Working stocks

After pipetting the pre-stocks (hint: use multi-channel pipette) we will now take aliquots from each pre-stock to form the working stock. Here is an example sheet used.

Pre-stock Source # [μL] [uL]
CoreP085 96 1 96 1 96
CoreP086 41 1 41 1 41
Neighbors 13 1 13 0 0
Polymers 13 1 13 0 0
ddH20 37 63
sum volume 200 200
Concentration 500 nM 500nM

Prepare Folding Reaction

Now that we have our working stock, we must decide whether to use 10 nM or 20 nM of our scaffold concentration, and whether hte final volume will be 50 μL or 100 μL. By default the FOBXM buffer solution and the MgCl2 magnesium screen will have already been prepared.