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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. After microwave for 2 minutes, swirl briefly and microwave for 1 minute more.
3. Slowly swirl in the ice bath.
4. Add ethidium bromid 6uL 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 70V for 2~3 hours.
8. Check image.

Atomic Force Microscopy (AFM)⁹

1. Apply ∼40 μL drop of 1×TAE/Mg++ and a ∼5 μL drop of annealed sample onto a freshly cleaved mica (left for about 2 minutes.)
cf) Diluted sample could be used to achieve the desired sample density. 
2. Add 1× TAE/8mM Ni++ to increase the strength of DNA-mica binding. 
3. To avoid bubbles, add additional ∼20 μL of 1× TAE/Mg++ buffer between the fluid cell and the AFM cantilever chip before placing the fluid cell on the mica puck.

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.