LabName:Experiment
Making DNA silk
Template preparation - Digestion process
Helper strand - 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 process
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 templates
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)
Circular DNA templates (50 nM), which were hybridized with Primer, were incubated with Φ29 DNA polymerase (1 unit/µL) at 30 ℃ for 4 h in the reaction buffer (50 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM MgCl2, 4 mM dithiothreitol, 200 µg/mL bovin serum albumin, 50 mM dNTP).
Making DNA Brick and Connector
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℃ at a rate of -1.0℃ for 1 minute.
Analysis
TBE-buffer
- Stock solution of EDTA
- Prepare a Stock Solution of 0.5 M EDTA for 500 mL
1. Weight 93.05g EDTA
2. Dissolve in 200 mL deionized water. (use magnetic stirrer before pH-titration)
3. NaOH in fume hood to pH 8 (Make sure that you have safety glasses on!). Add NaOH until solution becomes clear.
4. Adjust the volume to 500 mL with deionzed water.
- Stock solution of TBE
- Prepare a Stock Solution of TBE 10x TBE (1 liter)
1. Dissolve (use magnetic stirrer) 108 g Tris and 55 g Boric acid in 800 mL distilled water. (Start stirring directly after addition of magnetic stirrer to the tris/boric acid mix)
2. Add 40 mL 0.5 M Na2EDTA (pH 8.0) (alternatively use 9.3 g Na2EDTA)
3. Adjust volume to 1 Liter.
4. Store at room temperature. (Note : 10x TBE may take some time to dissolve, even with fast stirring)
5. Prepare a Working Solution of TBE
For agarose gel electrophoresis, TBE can be used at a concentration of 0.5x(1:10 dilution of the concentrated stock). Dilute the stock solution by 10x in deionized water.
Standard Gels3
- Large agarose gel(120 mL)
1. Measure 2.4 g for 2% gel agarose in 600 mL beaker.
2. Add 0.5x TBE up to 120 g.
3. Add dH2O up to 150 g.
4. Microwave on thigh for 2 minutes, swirl briefly, microwave additional 1 minute.
5. Pick up the beaker using a couple paper towels to provide insulation.
6. Gently swirl in an ice water bath until you no longer see steam rising form the beaker.
7. If running a MgCl2 gel, add 1 mL of 1.32 M MgCl2 (final concentration ~= 11mM). pipette down the side of the beaver to avoid creating bubbles.
8. Add 6 uL ethidium bromid (10 mg/mL) for concentration of 0.5 ug/mL
9. Gently swirl until EtBr is no longer visible.
10. Pour gel into casting tray, and insert comb with the thick teeth pointing down.
11. Set to 70 V, run for 2~3 hours before imaging
Atomic Force Microscopy (AFM)
AFM images were obtained using an MultiMode SPM with a Nanoscope IIIa controller (Veeco, Santa Barbara, CA) equipped with an Analog Q-control to optimize the sensitivity of the tapping mode (nanoAnalytics GmbH, Munster, Germany). 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. On a few occasions, supplemental 1× TAE/8mM Ni++ was added to increase the strength of DNA-mica binding (S4). 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. The AFM tips used were either the short and thin cantilever in the DNP-S oxide sharpened silicon nitride cantilever chip (Veeco Probes, Camarillo, CA) or the short cantilever in the SiNi chip (BudgetSensors, Sofia, Bulgaria).
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
2. Akinori, K., Makoto, K. (2007) Nucleic Acids Symposium Series Oxford Journals, 51, 331–332.
4. Biotech/Biomedical, Theresa Phillips, Mack TBE Buffer
5. Shih lab protocol
6. Peng Yin et al, (2008) science, 321, 824-826.
10. Addgene / Plasmid Reference > Restriction Digest of Plasmid DNA
New England Biolabs Inc. / Product-MscI
1. Jong Bum Lee et al, (2012) nature nanotechnology, 7, 816-820.
