LabName:Experiment
Making DNA silk
Template preparation - Digestion process
1. Select restriction enzymes to digest your plasmid. → MscI
- Heat Inactivation is 80℃ for 20min
- Note : To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence analysis program such as Addgene's Sequence Analyzer.
2. Determine an appropriate reaction buffer by reading the instructions for your enzyme. → 1X CutSmart™ Buffer
- 50 mM Potassium Acetate
- 20 mM Tris-acetate
- 10 mM Magnesium Acetate
- 100 μg/ml BSA
- pH 7.9 @ 25°C
3. 준비한 재료를 적절한 비율로 덜어 용기에 넣는다.
- Note : A typical restriction digestion reaction could look like this
- 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 1 hour.
6. To visualize the results of your digest, conduct gel electrophoresis.
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
Formation of the motifs was performed with 1µM DNA (for each strand) in a solution containing 40mM Tris, 20mM acetic acid, 2mM EDTA, and 12.5mM magnesium acetate (total volume was 100µL). This mixture was cooled from 90℃ to 25℃ at a rate of -1.0℃/min using a PCR thermal circler to anneal the strands.
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 Gels
- 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
- Small agarose gel (50 mL)
1. Small gels tend to be less photogenic than large ones.
2. Measure 0.5 g for 1% gel agarose in flask.
3. Add 0.5X TBE upto 50 g.
4. Microwave ~1 minute.
5. Cool to point where comfortable to touch bottom of flask.
6. Add 2.5 uL ethidium bromide (10 ug/mL) for final concentration of 0.5 ug/mL.
7. Set to 130 V, run for ~30 minutes before imaging.
Scanning Electron Microscope (SEM)
요거 필요 없을듯
The DNA hydrogel was placed onto the top of the SEM holder. The sample was metal-coated with Au/Pd.
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. Jong Bum Lee etc., (2012) nature nanotechnology, 7, 816-820.
2. Akinori, K., Makoto, K. (2007) Nucleic Acids Symposium Series Oxford Journals, 51, 331–332.
3. http://biotech.about.com/od/buffersandmedia/ht/MakeTBE.htm
4. https://lab.wyss.harvard.edu/104/index.php?title=Shih_Lab_Gel_Electrophoresis&oldid=32919
5. Peng Yin etc., (2008) science, 321, 824-826.
6. Addgene / Plasmid Reference > Restriction Digest of Plasmid DNA
7. New England Biolabs Inc. / Product-MscI
