LabName:Experiment: Difference between revisions

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| 1 || μl || 1 mM ATP || 50 μM
| 1 || μl || 1 mM ATP || 50 μM
|-
|-
| 1 || μl || 50 mM MnCl2 || 2.5 mM
| 1 || μl || 50 mM MnCl<sub>2</sub> || 2.5 mM
|-
|-
| 1 || μl || CircLigase ssDNA Ligase (100 U) || 5 U/μl
| 1 || μl || CircLigase ssDNA Ligase (100 U) || 5 U/μl
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:♦ CircLigase 10X Reaction Buffer: 0.5 M MOPS (pH 7.5) + 10 mM DTT + 0.1 M KCl + 50 mM MgCl2<br>
:♦ 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>
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>
3. Incubate the reaction for 10 minutes at 80°C to inactivate CircLigase ssDNA Ligase.<BR>
Line 72: Line 72:


===Rolling circle replication (RCR)<sup>5</sup>===
===Rolling circle replication (RCR)<sup>5</sup>===
1. Hybridize primer with Circular DNA (50 nM)<BR>
1. Prepare 50 nM circular DNA templates hybridized with primer.<br>
2. Add Φ29 DNA polymerase 1 unit/µL in 50 mM Tris-HCl, 50 mM dNTP, 10 mM MgCl<sub>2</sub>, 200 µg/mL bovin serum albumin, 10 mM (NH4)2SO<sub>4</sub>, and 4 mM dithiothreitol.<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.
3. Incubate at 30 ℃ during 4 h<BR>
4. Check how much reaction proceeds by electrophoresis.<BR>
: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.
<BR>


===Making DNA Brick and Connector<sup>6</sup>===
===Preparation of DNA Brick and Connector<sup>6</sup>===
1. Add 40mM Tris, 12.5mM magnesium acetate, 2mM EDTA, and 20mM acetic acid in solution with 1µM DNA. (total volume : 100µL)<BR>
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. Annealing from 90℃ to 25℃ (-1.0℃/min).<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<sup>7</sup>===
===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<sup>8</sup>===
===Standard Gels<sup>8</sup>===
* Agarose gel (120 mL)
• Agarose gel (120 mL)<BR>
1. Measure 2.4 g for 2% gel agarose.<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)<sup>9</sup>===
===Atomic Force Microscopy (AFM)<sup>9</sup>===
1. 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. <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>
2. On a few occasions, supplemental 1× TAE/8mM Ni++ was added to increase the strength of DNA-mica binding. <br>
cf) Diluted sample could be used to achieve the desired sample density. <BR>
3. 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. <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>


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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