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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. Hybridize primer with Circular DNA (50 nM)
2. Add Φ29 DNA polymerase 1 unit/µL in 50 mM Tris-HCl, 50 mM dNTP, 10 mM MgCl₂, 200 µg/mL bovin serum albumin, 10 mM (NH4)2SO₄, and 4 mM dithiothreitol.
3. Incubate at 30 ℃ during 4 h
4. Check how much reaction proceeds by electrophoresis.

It seemed appropriate to prepare 3~6 copies at maximum about 25kNT considering present length. Thus, We need for optimization by the condition including equivalence, concentration, reaction hour, etc.

Making DNA Brick and Connector⁶

1. Add 40mM Tris, 12.5mM magnesium acetate, 2mM EDTA, and 20mM acetic acid in solution with 1µM DNA. (total volume : 100µL)
2. Annealing from 90℃ to 25℃ (-1.0℃/min).

Analysis

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.