Biomod/2014/Folding: Difference between revisions
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30 µL sample was mixed with 5 µL 10X BlueJuice™ Gel Loading Buffer and loaded onto the gel. As a reference, 30µL p8064 scaffold (10nM) also with 5 µL 10X BlueJuice™ Gel Loading Buffer were applied. Furthermore, GeneRuler 1 kb Plus DNA Ladder was used as a marker.<br><br> | 30 µL sample was mixed with 5 µL 10X BlueJuice™ Gel Loading Buffer and loaded onto the gel. As a reference, 30µL p8064 scaffold (10nM) also with 5 µL 10X BlueJuice™ Gel Loading Buffer were applied. Furthermore, GeneRuler 1 kb Plus DNA Ladder was used as a marker.<br><br> | ||
The gel electrophoresis was carried out In TBE with 11mM MgCl<sub>2</sub> as running buffer | The gel electrophoresis was carried out In TBE with 11mM MgCl<sub>2</sub> as running buffer | ||
for 90 min.<br></font></p> | for 90 min.<br><br></font></p> | ||
<font size="+1pt">Gel extraction</font><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
The gel was examined under UV and the bands corresponding to successfully folded origami were cut. The DNA Origami solution was extracted through squeezing.<br><br></font></p> | |||
<font size="+1pt">Filtering</font><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
If the samples were not subjected to gel electrophoresis, filtering with an Amicon filter system (centrifugation 100k, 5 minutes, 10k rcf) was used to remove excess staple strands. The filtering was carried out 3 times with folding buffer.<br><br> | |||
The DNA origami solution was regained from the filter by centrifugation for 3 minutes at 1k rcf. | |||
<br><br></font></p> | |||
<font size="+1pt">Determining the optimal folding program</font><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
From the literature it is known that DNA origamis fold well when subjected to a thermal ramp,<sup>[1]</sup> cooling down the mixture of staple strands and scaffold from a high temperature (> 60 °C) to room temperature over a certain thermal ramp. For every new DNA origami, folding conditions have to be optimized. Therefore, we subjected the DNA origami folding mix containing all staples (100 nM each), scaffold p8064 (10 nM), 16 mM MgCl<sub>2</sub> in TE buffer to different folding programs. | |||
<br><br></font></p> | |||
<div align="center"><img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" ></div> | |||
<br> | |||
<i><font size="3 "><div align="center">Figure 1: Thermal ramps of the used folding programs.</font></i></div> | |||
<br><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
Afterwards, the samples were subjected to gel electrophoresis (Figure 2). | |||
<br><br></font></p> | |||
<div align="center"><img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" ></div> | |||
<br> | |||
<i><font size="3 "><div align="center">Figure 2: gel electrophoresis for testing different folding conditions. Lanes (from left): lane 1: 3DO; lane 2: 60HF; lane 3: 24HF; lane 4: p8064 scaffold as reference; lane 5: GeneRuler 1 kb Plus DNA Ladder as a marker.</font></i></div> | |||
<br><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
All three folding programs give rise to a sharp band. The sharp band in lane 4, which contains scaffold only, runs slower than the bands in the 1<sup>st</sup>-3<sup>rd<(/sup> lanes, which show the mobility of the DNA origami. Therefore we are certain that DNA origami folding took place in all three programs. Since the intensity is highest in lane 3, for all further experiments we used the folding program 24HF. | |||
<br><br></font></p> | |||
<font size="+1pt">Determining the optimal Mg<sup>2+</sup> concentration for folding</font><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
Since the folding is strongly dependent on the Mg<sup>2+</sup> concentration, we screened different Mg<sup>2+</sup> concentration for best folding efficiency. DNA origami folding mixtures with different Mg<sup>2+</sup> concentrations varying between 6 mM and 30 mM were subjected to the folding program 24HF and analyzed with gel electrophoresis, as shown in Figure 3.<br><br> | |||
Since the gel doesn’t show a clear tendency towards which concentration is best suited for the folding of the Nanoscooter, we further analyzed the samples with AFM. Figure 3 shows the difference between the Nanoscooter which was folded with the MgCl<sup>2</sup> concentrations of 6 and 18 mM. Obviously, the folding of the Origami with the latter concentration worked out best. | |||
<br><br></font></p> | |||
<div align="center"><img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" ></div> | |||
<br> | |||
<i><font size="3 "><div align="center">Figure 3: gel electrophoresis monitoring the folding process with different MgCl<sub>2</sub> conentrations 1st lane: GeneRuler 1 kb Plus DNA Ladder as a marker; 2<sup>nd</sup>-9<sup>th</sup> lane: variation of the MgCl<sub>2</sub> concentration; 10th lane: 8064 base pairs DNA scaffold as standard sample.</font></i></div> | |||
<br><br> | |||
<p align="justify"; style="line-height:2em"><font size="3pt"> | |||
Summing up, we successfully folded the new DNA origami and optimized the conditions. Best results are obtained by using a Mg<sup>2+</sup> concentration of 18 mM and the folding program 24HF. This now opens the way to use the Nanoscooter for all subsequent experiments. | |||
<br><br></font></p> | |||
<table> | |||
<tr><td><font size="3pt"><p align="justify"> | |||
[1]</font></td> <td><font size="3pt">P. Rothemund: <i>Folding DNA to create nanoscale shapes and patterns</i>, Nature,<b> 2006</b>, <i>440</i>, 297-302.</font></td></tr></table> | |||
</body> | </body> | ||
Revision as of 12:04, 24 October 2014
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DNA origami folding
DNA origamis were assembled with a tenfold excess of the staple strands with respect to the scaffold strand (1 pmol, p8064) in 1xTE buffer containing MgCl2 and using a thermocycler. Gel electrophoresis
0.75 g of agarose were added to 50 mL 0.5X TBE buffer and heated for 2 min in the microwave. After cooling down 2 µL Sybr® Safe DNA Gel Strain and 800 µL 1 M aqueous MgCl2 solution were added to the agarose gel. Gel extraction
The gel was examined under UV and the bands corresponding to successfully folded origami were cut. The DNA Origami solution was extracted through squeezing. Filtering
If the samples were not subjected to gel electrophoresis, filtering with an Amicon filter system (centrifugation 100k, 5 minutes, 10k rcf) was used to remove excess staple strands. The filtering was carried out 3 times with folding buffer. Determining the optimal folding program
From the literature it is known that DNA origamis fold well when subjected to a thermal ramp,[1] cooling down the mixture of staple strands and scaffold from a high temperature (> 60 °C) to room temperature over a certain thermal ramp. For every new DNA origami, folding conditions have to be optimized. Therefore, we subjected the DNA origami folding mix containing all staples (100 nM each), scaffold p8064 (10 nM), 16 mM MgCl2 in TE buffer to different folding programs.
<img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" >
Figure 1: Thermal ramps of the used folding programs.
Afterwards, the samples were subjected to gel electrophoresis (Figure 2).
<img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" >
Figure 2: gel electrophoresis for testing different folding conditions. Lanes (from left): lane 1: 3DO; lane 2: 60HF; lane 3: 24HF; lane 4: p8064 scaffold as reference; lane 5: GeneRuler 1 kb Plus DNA Ladder as a marker.
All three folding programs give rise to a sharp band. The sharp band in lane 4, which contains scaffold only, runs slower than the bands in the 1st-3rd<(/sup> lanes, which show the mobility of the DNA origami. Therefore we are certain that DNA origami folding took place in all three programs. Since the intensity is highest in lane 3, for all further experiments we used the folding program 24HF.
Determining the optimal Mg2+ concentration for folding
Since the folding is strongly dependent on the Mg2+ concentration, we screened different Mg2+ concentration for best folding efficiency. DNA origami folding mixtures with different Mg2+ concentrations varying between 6 mM and 30 mM were subjected to the folding program 24HF and analyzed with gel electrophoresis, as shown in Figure 3. <img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" >
Figure 3: gel electrophoresis monitoring the folding process with different MgCl2 conentrations 1st lane: GeneRuler 1 kb Plus DNA Ladder as a marker; 2nd-9th lane: variation of the MgCl2 concentration; 10th lane: 8064 base pairs DNA scaffold as standard sample.
Summing up, we successfully folded the new DNA origami and optimized the conditions. Best results are obtained by using a Mg2+ concentration of 18 mM and the folding program 24HF. This now opens the way to use the Nanoscooter for all subsequent experiments.
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