Biomod/2013/CUMC/Methods.html

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<!doctype html> Materials and Methods

Columbia University NanoMechanists

 

Materials and Methods

Materials

TRIS, EDTA, MgCl2, NaCl, CaCl were purchased from Fisher Scientific. Single stranded m13mp18 DNA strands were purchased from New England Biolabs. Single stranded oligo staples were designed through caDNAno and purchased through Integrated DNA Technologies. All sequences can be found in the supplementary section.

Preparation of Bundle Structure

Folding solution consisted of 20% 5X salt solution of EDTA, TRIS, NaCl, and MgCl2. Solution was thermally annealed from 56 degrees C to 44 degrees C at a rate of 0.2 degrees C/12 minutes. The structures were purified away from the excess of short staple using a 100kDa MWCO Millipore Amicon centrifugal filter spun down three rounds of 15 minutes each using three 500?l volumes folding buffer solution. The purified bundle structures were subsequently mixed with the inner barrel structure for assembly.

Preparation of Inner and Outer Barrel Structures

Folding solution consisted of 20% 5X salt solution of EDTA, TRIS, CaCl, and MgCl2. Solution was thermally annealed from 71 degrees C to 44 degrees C at a rate of 0.3 degrees C /8 minutes. The samples were likewise purified using a 100k Millipore filter spun down three rounds of 15 minutes each. The samples were then mixed post-fold to assemble.

Preparation of Post Fold Assembly

After structures were mixed they were heated at a constant rate of 37 degrees C for one hour. These samples were run on a 2% agarose gel to verify the folding, and purified using “freeze & squeeze” a commercial nucleic acid purification kit.

Preparation of Post Fold Assembly

After structures were mixed they were heated at a constant rate of 37 degrees C for one hour. These samples were run on a 2% agarose gel to verify the folding, and purified using “freeze & squeeze” a commercial nucleic acid purification kit.

Gel Electrophoresis

2% Agarose gels were used that consisted of MgCl2 and Ethidium Bromide for stain under UV imaging. Gels were run for two hours at 120V with a circulating pump maintain a uniform 6°C buffer solution.

TEM Imaging

TEM images were taken with a negative stain. All purified samples imaged using TEM imaging were diluted to a 4 nM concentration.

Stojanovic Research Group
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