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='''Making DNA silk'''=
='''Making DNA silk'''=
===Template preparation - Digestion process===
===Template preparation - Digestion process===
[[Image:141025KMG_MscI.png|200px]]<BR>
'''Helper strand<sup>1,2</sup>''' - The circular single-stranded DNA is combined with helper strands.<BR>
1. Select restriction enzymes to digest your plasmid. → MscI<BR>
1. This could be achieved by taking the equivalent of well-calibrated pipette and combining a 5㎕ drop of solution from each tubes. <BR>
:Heat Inactivation is 80℃ for 20min
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).<BR>
: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.
3. The mixture of strands is heated to near boiling(90℃) and cooled to room temperature(20℃) over about 2 hours.<BR>
2. Determine an appropriate reaction buffer by reading the instructions for your enzyme. → 1X CutSmart™ Buffer<BR>
<br>
 
'''Digestion process<sup>3</sup>'''<BR>
1. Choose restriction enzymes to digest plasmid.<BR>
:⇒ MscI(Heat Inactivation is 80℃ for 20 min)<br>
:[[Image:141025KMG_MscI.png|200px]]<BR>
:♦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.<BR>
:⇒ 1X CutSmart™ Buffer <BR>
:100 μg/ml BSA
:50 mM Potassium Acetate
:50 mM Potassium Acetate
:10 mM Magnesium Acetate
:20 mM Tris-acetate
:20 mM Tris-acetate
:10 mM Magnesium Acetate
:pH 7.9(at 25°C)
:100 μg/ml BSA
3. Put the appropriate proportion of prepared material on container.<BR>
:pH 7.9 @ 25°C
3. 준비한 재료를 적절한 비율로 덜어 용기에 넣는다.<BR>
:Note : A typical restriction digestion reaction could look like this
:1μg M13mp18
:1μg M13mp18
:0.2 μL MscI (R0534L)
:0.2 μL MscI (R0534L)
:x μL 1X CutSmart™ Buffer (to bring total volume to 50 μL)
:x μL 1X CutSmart™ Buffer (to bring total volume to 50 μL)
4. Mix gently by pipetting.<BR>
4. Mix gently by pipetting.<BR>
5. Incubate tube at 37°C for 1 hour.<BR>
5. Incubate tube at 37°C for an hour.<BR>
6. To visualize the results of your digest, conduct gel electrophoresis.<BR>
6. Results of a digest could be visualized by conducting gel electrophoresis.<BR>
<BR>
<BR>


===Rolling circle replication (RCR)===
===Circularizing templates<sup>4</sup>===
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).<BR>
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).<br>
 
1. Combine the following reaction components:
 
::{| class="wikitable"
|-
!  !! 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 MnCl<sub>2</sub> || 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 MgCl<sub>2</sub><br>
2. Incubate the reaction at 60°C for an hour.<br>
3. Incubate the reaction for 10 minutes at 80°C to inactivate CircLigase ssDNA Ligase.<BR>
<BR>
<BR>


===Making DNA Brick and Connector===
===Rolling circle replication (RCR)<sup>5</sup>===
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.<BR>
1. Prepare 50 nM circular DNA templates hybridized with primer.<br>
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 Connector<sup>6</sup>===
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)<BR>
2. To anneal the strands, cool this mixture from 90℃ to 25℃ using PCR thermal circler (-1.0℃/min).<BR>
<BR>
<BR>


='''Analysis'''=
='''Analysis'''=
===TBE-buffer===
===TBE-buffer<sup>7</sup>===
* Stock solution of EDTA
* TBE-buffer
- Prepare a Stock Solution of 0.5 M EDTA for 500 mL<BR>
- Prepare a Stock Solution of 500mM EDTA (500 mL)<br>
1. Weight 93.05g EDTA<BR>
1. Measure EDTA 93.05g<br>
2. Dissolve in 200 mL deionized water. (use magnetic stirrer before pH-titration)<BR>
2. Dissolve in 0.2L deionized water (by using magnetic bar).<br>
3. NaOH in fume hood to pH 8 (Make sure that you have safety glasses on!). Add NaOH until solution becomes clear.<BR>
3. NaOH in hood up to pH 8 (dedicate to safety). Add NaOH up to solution becomes clear.<br>
4. Adjust the volume to 500 mL with deionzed water.<BR>
4. Change the volume to 0.5 L by using deionzed water.<br>
<BR>
<BR>
* Stock solution of TBE<BR>
* Stock solution of TBE<BR>
- Prepare a Stock Solution of TBE 10x TBE (1 liter)<BR>
- Prepare a Stock Solution of TBE 10x TBE (1 liter)<BR>
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)<BR>
1. Dissolve 108g Tris and 55g Boric acid in 800mL of distilled water using magnetic stirrer.<BR>
2. Add 40 mL 0.5 M Na2EDTA (pH 8.0) (alternatively use 9.3 g Na2EDTA)<BR>
2. Add 0.5 M Na<sub>2</sub>EDTA(pH 8.0) 40mL<BR>
3. Adjust volume to 1 Liter.<BR>
3. Adjust volume to 1 Liter.<BR>
4. Store at room temperature. (Note : 10x TBE may take some time to dissolve, even with fast stirring)<BR>
4. Store at room temperature.<BR>
5. Prepare a Working Solution of TBE<BR>
5. Prepare a Working Solution of TBE<BR>
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.<BR>
<BR>


===Standard Gels===
===Standard Gels<sup>8</sup>===
* Large agarose gel(120 mL)
• Agarose gel (120 mL)<BR>
1. Measure 2.4 g for 2% gel agarose in 600 mL beaker.<BR>
1. Add 2% agarose gel 2.4 g and 0.5x TBE 120 g, dH2O 150 g in beaker.<BR>
2. Add 0.5x TBE up to 120 g.<BR>
2. After microwave for 2 minutes, swirl briefly and microwave for 1 minute more.<BR>
3. Add dH2O up to 150 g.<BR>
3. Slowly swirl in the ice bath.<BR>
4. Microwave on thigh for 2 minutes, swirl briefly, microwave additional 1 minute.<BR>
4. Add ethidium bromid 6uL for concentration of 0.5 ug/mL.<BR>
5. Pick up the beaker using a couple paper towels to provide insulation.<BR>
5. Gently swirl until ethidium bromid is no longer visible.<BR>
6. Gently swirl in an ice water bath until you no longer see steam rising form the beaker.<BR>
6. Pour gel into a casting tray, and insert comb.<BR>
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.<BR>
7. Set to 70V for 2~3 hours.<BR>
8. Add 6 uL ethidium bromid (10 mg/mL) for concentration of 0.5 ug/mL<BR>
8. Check image.<BR>
9. Gently swirl until EtBr is no longer visible.<BR>
10. Pour gel into casting tray, and insert comb with the thick teeth pointing down.<BR>
11. Set to 70 V, run for 2~3 hours before imaging<BR>
<BR>
<BR>


===Atomic Force Microscopy (AFM)===
===Atomic Force Microscopy (AFM)<sup>9</sup>===
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).<BR>
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.)<BR>
cf) Diluted sample could be used to achieve the desired sample density. <BR>
2. Add 1× TAE/8mM Ni++ to increase the strength of DNA-mica binding. <BR>
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.<BR>
<BR>
<BR>


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

Latest revision as of 23:51, 25 October 2014

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Making DNA silk

Template preparation - Digestion process

Helper strand1,2 - 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.

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