Biomod/2014/Kansai/Protocols: Difference between revisions
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We observed this product by AFM. | We observed this product by AFM. AFM imaging of DNA origami was performed on a Multimode 8/ Nanoscope system (Bruker AVS). | ||
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''' | '''Self assembly of DNA origami'''<br> | ||
The formation of DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staples DNA (20 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg2+ buffer, 50 µL). This mixture was cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands. | The formation of DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staples DNA (20 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg2+ buffer, 50 µL). This mixture was cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands. | ||
''' | '''AFM observation'''<br> | ||
The mixture (1 µL) was deposited on freshly cleaved mica, additional 1X TAE/Mg2+ buffer (40 µL) was added, and the imaging was performed in the fluid Tapping mode with a BL-AC40TS tip (Olympus, Japan). | |||
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Revision as of 00:26, 29 August 2014
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Each staple DNA were dissolved by sterilized water that the concentration of each solution might be 100 μM.
These solutions were taken 2 μL each and mixed.
The volume of the solution was diluted with sterilized water to 500μL. we prepared staple DNA mixture.The sample (50 μM) was prepared mixing M13mp18 (4nM), staple DNA mixture (20nM) and 1×TAE/Mg2+ (12.5 mM). This mixture was kept at 90˚C for 10 minutes and cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands.
The annealed mixture (1μL) was dripped on mica and added 1×TAE/Mg2+ (12.5 mM).
We observed this product by AFM. AFM imaging of DNA origami was performed on a Multimode 8/ Nanoscope system (Bruker AVS).
Protocols
・Materials
Staples DNA were purchased from IDT ( U.S.A )
Self assembly of DNA origami
The formation of DNA origami was performed with M13mp18 ssDNA (4 nM, Takara, Japan), staples DNA (20 nM for each strand) in a solution containing Tris (40 mM), acetic acid (20 mM), EDTA (10 mM), and magnesium acetate (12.5 mM, 1 X TAE/Mg2+ buffer, 50 µL). This mixture was cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands.
AFM observation
The mixture (1 µL) was deposited on freshly cleaved mica, additional 1X TAE/Mg2+ buffer (40 µL) was added, and the imaging was performed in the fluid Tapping mode with a BL-AC40TS tip (Olympus, Japan).