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Making DNA silk

Template preparation - Digestion process

Helper strand^1,2 - The circular single-stranded DNA is combined with helper strands.
1. This could be achieved by taking the equivalent of well-calibrated pipette and combining a 5㎕ drop of solution from each tubes.
2. After the combination of the scaffold and helper strands, a slight buffer(to control pH) and magnesium salt are added.(Magnesium Mg++ ion neutralizes negative charge of DNA and allow single-stranded DNA to gather and form the double helix).
3. The mixture of strands is heated to near boiling(90℃) and cooled to room temperature(20℃) over about 2 hours.

Digestion process³
1. Choose restriction enzymes to digest plasmid.

⇒ MscI(Heat Inactivation is 80℃ for 20 min)

♦Note: To determine proper restriction enzymes to cut DNA sequence (and where they should be cut), could get some help from a sequence analysis program like Addgene's Sequence Analyzer.

2. Select a proper reaction buffer by checking the instructions of enzyme.

⇒ 1X CutSmart™ Buffer

100 μg/ml BSA

50 mM Potassium Acetate

10 mM Magnesium Acetate

20 mM Tris-acetate

pH 7.9(at 25°C)

3. Put the appropriate proportion of prepared material on container.

1μg M13mp18

0.2 μL MscI (R0534L)

x μL 1X CutSmart™ Buffer (to bring total volume to 50 μL)

4. Mix gently by pipetting.
5. Incubate tube at 37°C for an hour.
6. Results of a digest could be visualized by conducting gel electrophoresis.

Circularizing templates⁴

Storage: Store at –20°C in a freezer. Storage Buffer: CircLigase ssDNA Ligase is prepared in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 100 mM NaCl, 0.1% Triton® X-100 and 1 mM dithiothreitol(DTT).

1. Combine the following reaction components:

	Unit		Final Concentration
10	μl	Sterile water	---
2	pmol	Single-stranded DNA template	0.5 pmol/μl
1	μl	CircLigase 10X Reaction Buffer	1X
1	μl	1 mM ATP	50 μM
1	μl	50 mM MnCl2	2.5 mM
1	μl	CircLigase ssDNA Ligase (100 U)	5 U/μl
20	μl	Total reaction volume

♦ CircLigase 10X Reaction Buffer: 0.5 M MOPS (pH 7.5) + 10 mM DTT + 0.1 M KCl + 50 mM MgCl₂

2. Incubate the reaction at 60°C for an hour.
3. Incubate the reaction for 10 minutes at 80°C to inactivate CircLigase ssDNA Ligase.

Rolling circle replication (RCR)⁵

1. Prepare 50 nM circular DNA templates hybridized with primer.
2. Incubated with Φ29 DNA polymerase(1unit/µL) in the reaction buffer composed of 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM dithiothreitol, 200 µg/mL BSA(bovin serum albumin) and 50 mM dNTP at 30 ℃ for 4 hours.

Preparation of DNA Brick and Connector⁶

1. Add 1µM DNA in a solution compose of 40mM Tris, 12.5mM magnesium acetate, 2mM EDTA, and 20mM acetic acid.(Total volume should be 100µL)
2. To anneal the strands, cool this mixture from 90℃ to 25℃ using PCR thermal circler (-1.0℃/min).

Analysis

TBE-buffer⁷

• TBE-buffer

- Prepare a Stock Solution of 500mM EDTA (500 mL)
1. Measure EDTA 93.05g
2. Dissolve in 0.2L deionized water (by using magnetic bar).
3. NaOH in hood up to pH 8 (dedicate to safety). Add NaOH up to solution becomes clear.
4. Change the volume to 0.5 L by using deionzed water.

• Stock solution of TBE
  

- Prepare a Stock Solution of TBE 10x TBE (1 liter)
1. Dissolve 108g Tris and 55g Boric acid in 800mL of distilled water using magnetic stirrer.
2. Add 0.5 M Na2EDTA(pH 8.0) 40mL
3. Adjust volume to 1 Liter.4. Store at room temperature.5. Prepare a Working Solution of TBE

Standard Gels⁸

• Agarose gel (120 mL)

1. Add 2% agarose gel 2.4 g and 0.5x TBE 120 g, dH2O 150 g in beaker.
2. Microwave (2 minutes), swirl briefly, and microwave (1 minute).
3. Slowly swirl in an ice bath.
4. Add ethidium bromid 6 uL for concentration of 0.5 ug/mL.
5. Gently swirl until ethidium bromid is no longer visible.
6. Pour gel into a casting tray, and insert comb.
7. Set to 70 V for 2~3 hours.
8. Check image.

Atomic Force Microscopy (AFM)⁹

1. A ∼40 μL drop of 1× TAE/Mg++ followed by a ∼5 μL drop of annealed sample was applied onto the surface of a freshly cleaved mica and left for approximately 2 minutes. Sometimes, additional dilution of the sample was performed to achieve the desired sample density.
2. On a few occasions, supplemental 1× TAE/8mM Ni++ was added to increase the strength of DNA-mica binding.
3. Before placing the fluid cell on top of the mica puck, an additional ∼20 μL of 1× TAE/Mg++ buffer was added to the cavity between the fluid cell and the AFM cantilever chip to avoid bubbles.
1. Agarose gel electrophoresis
2. Atomic Force Microscopy (AFM)

Reference

1. Design of DNA origami / aul W.K. Rothemund / California Institute of Technology, Pasadena, CA 91125
2. Addgene / Plasmid Reference > Restriction Digest of Plasmid DNA
3. New England Biolabs Inc. / Product-MscI
4. epicentre an illumina company – CirLigase ssDNA Ligase / Cat.Nos. CL4111K and 4115K / Lit. # 222 • 10/2012 1EPILIT222 Rev. A
5. Jong Bum Lee et al, (2012) nature nanotechnology, 7, 816-820.
6. Akinori, K., Makoto, K. (2007) Nucleic Acids Symposium Series Oxford Journals, 51, 331–332.
7. Biotech/Biomedical, Theresa Phillips, Mack TBE Buffer
8. Shih lab protocol
9. Peng Yin et al, (2008) science, 321, 824-826.