Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/Protocols: Difference between revisions
| Line 20: | Line 20: | ||
of the template was unknown. Primer sequences are listed in Table A.2. The | of the template was unknown. Primer sequences are listed in Table A.2. The | ||
buffer and polymerase was from Invitrogen. The PCR machine used was a VWR | buffer and polymerase was from Invitrogen. The PCR machine used was a VWR | ||
Collection DOPPIO Thermal Cycler. The program was 27 cycles of; | Collection DOPPIO Thermal Cycler. The program was 27 cycles of; 94°C for 20 seconds, | ||
55°C for 20 seconds and 72°C for 20 seconds. The ramp time was 3°C/s and | |||
the PCR machine stored the mixture at | the PCR machine stored the mixture at 10°C. The PCR product was purified using | ||
a illustra GFXTM PCR DNA and Gel Band Purification Kit from GE Healthcare and | a illustra GFXTM PCR DNA and Gel Band Purification Kit from GE Healthcare and | ||
following the provided procedure. 500 μl Capture buffer type 3 was added to the | following the provided procedure. 500 μl Capture buffer type 3 was added to the | ||
| Line 41: | Line 41: | ||
the purified PCR product, 1X FastDigest | the purified PCR product, 1X FastDigest | ||
R buffer, 1X EcoRI, 1X BamHI was | R buffer, 1X EcoRI, 1X BamHI was | ||
incubated at | incubated at 37°C for 30 minutes. Another mixture with a total volume of 20 μl | ||
containing 1 μg pUC18, 1X FastDigest | containing 1 μg pUC18, 1X FastDigest | ||
R buffer, 1X EcoRI, 1X BamHI was incubated | R buffer, 1X EcoRI, 1X BamHI was incubated | ||
at | at 37°C for 30 minutes. The digested PCR product and plasmid was purified following | ||
the procedure explained earlier for the GFXTM Purification Kit. | the procedure explained earlier for the GFXTM Purification Kit. | ||
The enzyme and buffer used for the ligation were from Fermantas. The reaction | The enzyme and buffer used for the ligation were from Fermantas. The reaction | ||
was carried out in a total volume of 20 μl by mixing 250 ng digested pUC18, | was carried out in a total volume of 20 μl by mixing 250 ng digested pUC18, ~4- | ||
fold excess of digested PCR product, 1X T4 DNA Ligase buffer, 1X T4 DNA Ligase | fold excess of digested PCR product, 1X T4 DNA Ligase buffer, 1X T4 DNA Ligase | ||
(200 CEU/μl) and incubating at | (200 CEU/μl) and incubating at 17°C over night. | ||
===Transfection of cells=== | ===Transfection of cells=== | ||
| Line 56: | Line 56: | ||
Prepared heat-shock cells (E. coli XL1-blue from Stratagen) was transfected by adding | Prepared heat-shock cells (E. coli XL1-blue from Stratagen) was transfected by adding | ||
5 μl of the ligation product to 200 μl cells and incubated on ice for 40 min. The | 5 μl of the ligation product to 200 μl cells and incubated on ice for 40 min. The | ||
cells were heat-shocked at | cells were heat-shocked at 42°C for 100 seconds and incubated on ice for 5 min. | ||
After the addition of 800 μl of LB media the mixture was incubated at | After the addition of 800 μl of LB media the mixture was incubated at 37°C for | ||
an hour. The cells were then spun down for 30 sec. at 12,000 rpm and | an hour. The cells were then spun down for 30 sec. at 12,000 rpm and ~900 μl of | ||
the supernatant was removed. After resuspension the cells were diluted 1. | the supernatant was removed. After resuspension the cells were diluted 1.000x | ||
and plated on agar and incubate at | and plated on agar and incubate at 37°C over night. Cells were then replated and | ||
incubated at | incubated at 37°C over night. | ||
The agar plates were made by mixing 10 g Typtone, 5 g yeast extract, 10 g NaCl, | The agar plates were made by mixing 10 g Typtone, 5 g yeast extract, 10 g NaCl, | ||
15 g Agar and adding double distilled water to a final volume of one liter. The | 15 g Agar and adding double distilled water to a final volume of one liter. The | ||
mixture was autoclaved and cooled to | mixture was autoclaved and cooled to 55°C and X-gal and Ampicillin were added | ||
to a final concentration of 20 μg/ml and 50 mg/ml, respectively. | to a final concentration of 20 μg/ml and 50 mg/ml, respectively. | ||
Cells were transfered to a tube that contained 5 ml LB media and Ampicillin | Cells were transfered to a tube that contained 5 ml LB media and Ampicillin | ||
(100 mg/ml), using a autoclaved toothpick. The solution was incubated at | (100 mg/ml), using a autoclaved toothpick. The solution was incubated at 37°C | ||
over night while shaking at 200 rpm. The cells where then spun down for 2 min. at | over night while shaking at 200 rpm. The cells where then spun down for 2 min. at | ||
7.000 rcf and the media was removed. | 7.000 rcf and the media was removed. | ||
| Line 111: | Line 111: | ||
manufacturers procedure was followed. A mixture with a total volume of 20 μl was | manufacturers procedure was followed. A mixture with a total volume of 20 μl was | ||
made, containing 1X MEGAscript Enzyme Mix (T7), 1X Reaction Buffer, 7.5mM | made, containing 1X MEGAscript Enzyme Mix (T7), 1X Reaction Buffer, 7.5mM | ||
NTP mix (T7) and template with an expected concentration of | NTP mix (T7) and template with an expected concentration of ~1 μM. The content | ||
was mixed thoroughly and incubated at | was mixed thoroughly and incubated at 37°C over night. The template was | ||
removed by adding 1X TURBO DNase (2 U/μl) and incubating at | removed by adding 1X TURBO DNase (2 U/μl) and incubating at 37°C for 15 min. | ||
===Purification of RNA scaffold=== | ===Purification of RNA scaffold=== | ||
| Line 134: | Line 134: | ||
then presipitated by transferring the supernatant to a new effendorf tube containing | then presipitated by transferring the supernatant to a new effendorf tube containing | ||
750 μl 96% ethanol. The mixture was vortexed for 30 sec. and then put on dry | 750 μl 96% ethanol. The mixture was vortexed for 30 sec. and then put on dry | ||
ice for 15 min. After centrifuging at | ice for 15 min. After centrifuging at 4°C for 15 min. at 14.000 rcf, the supernatant | ||
was discarded and 450 μl 70% ethanol was added. After centrifuging again at | was discarded and 450 μl 70% ethanol was added. After centrifuging again at 4°C | ||
for 15 min. at 14.000 rcf, the supernatant was removed completely and the pellet | for 15 min. at 14.000 rcf, the supernatant was removed completely and the pellet | ||
was resuspended in 20 μl RNase-free water. The concentration was measured by | was resuspended in 20 μl RNase-free water. The concentration was measured by | ||
Revision as of 14:57, 1 November 2011
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</style> <body> <a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists"> <IMG class="displogo" src="http://openwetware.org/images/6/67/DNALogo.png" alt=""/> </a> <script type='text/javascript' src='https://ajax.googleapis.com/ajax/libs/jquery/1.6.4/jquery.min.js'></script> <script> function mainmenu(){ $(" #nav li").hover(function(){ $(this).find('ul:first').css({visibility: "visible",display: "none"}).show(400); },function(){ $(this).find('ul:first').css({visibility: "hidden"}); }); }
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<ul id="nav">
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team">Team</a>
<ul>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/Mie">Mie</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/Irene">Irene</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/Jens">Jens</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/HansChristian">Hans Christian</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/Steffen">Steffen</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/OompaLoompas">Oompa-Loompas</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Team/Acknowledgements">Acknowledgements</a></li>
</ul>
</li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Idea">Idea</a>
<ul>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Idea#Idea">Video</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Idea#Goals">Goals</a></li>
</ul>
</li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project">Project</a>
<ul>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project#Self-assembly_and_characterization_of_the_structure">Self-assembly of the structure</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project#Gene_knockdown_using_the_RNAi_pathway">Gen knock-down using the RNAi pathway</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project#Controlled_opening_of_the_structure">Controlled opening of the structure</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods">Methods ></a>
<ul>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods#Gel_electrophoresis">Gels electrophoresis</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods#SAXS">SAXS</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods#FRET">FRET</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods#Dicer">Dicer</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Methods#Dual_Luciferace_assay">Dual Luciferase assay</a></li>
</ul>
</li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Project/Abbreviations">Abbreviations</a></li>
</ul>
</li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Overview">Overview</a>
<ul>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Overview/Goals">Goals</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Overview/Achievements">Achievements</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Overview/FutureWork">Future work</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Overview/Perspectives">Perspectives</a></li>
</ul>
</li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary">Supplementary</a>
<ul>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/Protocols">Protocols</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/Sequences">Sequences</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/SAXS">SAXS</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/FRET">FRET</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/Luciferase">Luciferase</a></li>
<li><a href="http://openwetware.org/wiki/Biomod/2011/Aarhus/DanishNanoArtists/Supplementary/Literature">Literature</a></li>
</ul>
</li>
</ul>
</body> </html>
Sequencing and transcription of the scaffold
To be able to sequence the entire scaffold the template DNA was first amplified with PCR and then cloned into a pUC18 vector. The transcription of scaffold was done using a PCR product of the template DNA.
Amplification of DNA template
PCR was used to amplify the DNA sequence used as template in the transcription of the RNA scaffold. The template for the PCR was a plasmid used for Luciferaseassays, kindly provided by Jesper B. Bramsen. A mixture with a final volume of 100 μl was made containing 1X Taq Reaction buffer (200mM Tris pH 8.4, 500mM KCl), 2.5mM MgCl2, 200μM dNTP mix, 200nM forward primer (FW-Luc-T7-EcoRI), 200nM backward primer (BW-Luc-BamHI), 1μM Taq DNA polymerase, 1 μl template (genomic siCheck2 siEGFP1 with an unknown concentration). The concentration of the template was unknown. Primer sequences are listed in Table A.2. The buffer and polymerase was from Invitrogen. The PCR machine used was a VWR Collection DOPPIO Thermal Cycler. The program was 27 cycles of; 94°C for 20 seconds, 55°C for 20 seconds and 72°C for 20 seconds. The ramp time was 3°C/s and the PCR machine stored the mixture at 10°C. The PCR product was purified using a illustra GFXTM PCR DNA and Gel Band Purification Kit from GE Healthcare and following the provided procedure. 500 μl Capture buffer type 3 was added to the 100 μl sample and vortexed. The mixture was added to GFX MicroSpinTM column, spun for 30 seconds at 16.000 rcf and the flow through was discarded. 500 μl Wash buffer type 1 was added to the column, spun for 30 seconds at 16.000 rcf and the collection tube was discarded. 50 μl Elution buffer type 6 (nuclease-free water) was added to the column, which was inserted into a clean eppendorf tube. After one minute the column was spun for one minute at 16.000 rcf, eluding the template into the eppendorf tube.
Digestion and ligation of plasmid and PCR product
The purified PCR product was digested with the restriction enzymes EcoRI and BamHI. Fermentas FastDigest R enzymes were used and the procedure provided by the manufacturer was followed. A mixture with a total volume of 35 μl containing the purified PCR product, 1X FastDigest R buffer, 1X EcoRI, 1X BamHI was incubated at 37°C for 30 minutes. Another mixture with a total volume of 20 μl containing 1 μg pUC18, 1X FastDigest R buffer, 1X EcoRI, 1X BamHI was incubated at 37°C for 30 minutes. The digested PCR product and plasmid was purified following the procedure explained earlier for the GFXTM Purification Kit.
The enzyme and buffer used for the ligation were from Fermantas. The reaction was carried out in a total volume of 20 μl by mixing 250 ng digested pUC18, ~4- fold excess of digested PCR product, 1X T4 DNA Ligase buffer, 1X T4 DNA Ligase (200 CEU/μl) and incubating at 17°C over night.
Transfection of cells
Prepared heat-shock cells (E. coli XL1-blue from Stratagen) was transfected by adding 5 μl of the ligation product to 200 μl cells and incubated on ice for 40 min. The cells were heat-shocked at 42°C for 100 seconds and incubated on ice for 5 min. After the addition of 800 μl of LB media the mixture was incubated at 37°C for an hour. The cells were then spun down for 30 sec. at 12,000 rpm and ~900 μl of the supernatant was removed. After resuspension the cells were diluted 1.000x and plated on agar and incubate at 37°C over night. Cells were then replated and incubated at 37°C over night.
The agar plates were made by mixing 10 g Typtone, 5 g yeast extract, 10 g NaCl, 15 g Agar and adding double distilled water to a final volume of one liter. The mixture was autoclaved and cooled to 55°C and X-gal and Ampicillin were added to a final concentration of 20 μg/ml and 50 mg/ml, respectively.
Cells were transfered to a tube that contained 5 ml LB media and Ampicillin (100 mg/ml), using a autoclaved toothpick. The solution was incubated at 37°C over night while shaking at 200 rpm. The cells where then spun down for 2 min. at 7.000 rcf and the media was removed.
Purification of ligation product
GeneJETTM Plasmid Miniprep Kit from Fermentas was used to purify the ligation product from the cells. The procedure provided by Fermentas was as followed. The pelleted cells were resuspended in 250 μl Resuspension Solution (containing RNase A) by vortexing. The solution was mixed with 250 μl Lysis Solution by inversion, followed by the addition of 350 μl Neutralization Solution and then centrifuged for 5 min at 13.000 rcf. The supernatant was loaded on the GeneJETTM spin column and centrifuged for 1 min. at 13.000 rcf. The column was washed two times by adding 500 μl Wash Solution, centrifuging for 1 min. at 13.000 rcf and discarding the flow-through. After the second wash the column was centrifuged for 1 min. more at 13.000 rcf. The coulmn was transfered to a clean eppendorf tube, 50 μl Elution Buffer was added and after 2 min. the column was spun for 2 min. at 13.000 rcf, eluding the purified ligation product.
Sequencing
BigDye® Terminator v3.1 Cycle Sequencing Kit was used and the procedure provided by the manufacturer was followed. Two reactions were performed, one in each direction, to ensure higher fidelity in the analysis of the sequencing. The sequencing mixture contained 200 ng plasmid DNA, 5 pmol primer (only forward or backward), 1X BigDye Sequencing Buffer, 1X BigDye Ready Reaction Premix. The primers used were standard sequencing primers ’FW M13 (-20)’ and ’BW M13’ (see the sequences in Table A.2). Same machine and program as for PCR was used for the sequencing reaction. After the reaction water was added to 20 μl and the mixture was delivered to Hans Hjorth who purified and sequenced it. CLC Main Workbench was used to analyze the sequences.
In vitro transcription
The template for the transcription of the scaffold was a PCR product with a T7 promoter incorporated upstream of the scaffold sequence. The PCR was carried out as the amplification procedure explained earlier, but the primers used were ’FW-Luc-T7-EcoRI’ and ’BW-Luc’. See sequences in Table A.2.
MEGAscript™ High Yield Transcription Kit from Ambion was used and the manufacturers procedure was followed. A mixture with a total volume of 20 μl was made, containing 1X MEGAscript Enzyme Mix (T7), 1X Reaction Buffer, 7.5mM NTP mix (T7) and template with an expected concentration of ~1 μM. The content was mixed thoroughly and incubated at 37°C over night. The template was removed by adding 1X TURBO DNase (2 U/μl) and incubating at 37°C for 15 min.
Purification of RNA scaffold
A denaturing 6% polyacrylamide gel (PAG) was made, with SequaGelTM Urea- Gel System from National Diagnostics by mixing 1X TBE buffer, 1X Concentrate, 1X Diluent, 2.5μM Tetramethylethylenediamine (TEMED), 7μM Ammonium persulfate (APS) in a total volume of 40 ml and casting the gel between two glass plates. The gel was pre-run for a total of 25 min. at 15Wand the wells were cleaned, before loading the transcription product. The gel was run for 45 min. at 15 W and the band of interest was cut out using a scalpel. The cut-out was transfered to an elution buffer containing 330 μl TE buffer with 200mM NaOAc and 100 μl phenol (pH 6.6). The elution mixture was set to shake at 800 rpm over night. After elution 200 μl phenol was added to the mixture, which was vortexed for 30 sec. and spun at 14.000 rcf for 2 min. The following three step were all vortexed and spun in the same way. 1) The supernatant was transfered to a new effendorf tube containing 300 μl phenol. 2) The supernatant was transfered to a new effendorf tube containing 150 μl phenol and 150 μl chloroform. 3) The supernatant was transfered to a new effendorf tube containing 300 μl chloroform. The RNA was then presipitated by transferring the supernatant to a new effendorf tube containing 750 μl 96% ethanol. The mixture was vortexed for 30 sec. and then put on dry ice for 15 min. After centrifuging at 4°C for 15 min. at 14.000 rcf, the supernatant was discarded and 450 μl 70% ethanol was added. After centrifuging again at 4°C for 15 min. at 14.000 rcf, the supernatant was removed completely and the pellet was resuspended in 20 μl RNase-free water. The concentration was measured by OD260.
Quality control of staple strands
The staples were run on a gel with the scaffold to determine weather or not they were contaminated with RNase. The FRET staples were further tested by measuring the absorption and thereby determining the concentration of fluorophores compare to the concentration of DNA.
Gel electrophoresis
A 1.5% agarose gel was made by mixing 0.9 g UltraPureTM Agrose from Invitrogen and 0.5X TBE in a total volume of 60 ml. The solution was heated in the microwave oven until the agarose was dissolved. After the solution had cooled, 1X SYBR® Safe was added and the solution was poured into the mold and cast using a comb with twelve teeth. The gel was electrophoresed for 20 minutes at 120 V in 0.5X TBE. ’Native’ agarose gels were also used, with the only difference being the addition of 5 mM MgAc to the gel and buffer as well as a change in the electrophoresis conditions (2 hours in 5�C at 100 V). A Typhoon TRIO+TM Variable Mode Imager was used to visualize the gels. The excitation was at 532 nm and the emission filter was 580 BP 30.
Absorption measurements
Absorption measurements were used to determine the quality of the FRET oligos, by calculating the amount of DNA in a sample compared with the amount of fluorophores based on the absorbance. A 2μM solution of each of the four FRET oligos (sequences listed in Table A.2) were made using nuclease-free water as solvent. 65 μl was pipetted to a quartz cuvette with an optical path length of 3mm. A cuvette with nuclease-free water was used as reference. The spectrophotometer used was a Shimadzu UV-3600 UV-VIS-NIR and the spectra were recorded from 200nm to 800 nm.
Self-assembly procedures
The S structure was self-assembled by the following procedure. The staples were first treated with SDS by mixing 10 μM of each staple strand, 1% SDS and 63 mM Tris pH 7.5 in a total volume of 50 μl. The mixture was heated to 95�C for 2 minutes. Then scaffold, TAE buffer and MgAc was added so the final concentrations were 0.686 μM scaffold, 1X TAE, 12.5 mM MgAc with a total volume of 70 μl.
The F structures were self-assembled by the following procedure. The staples were first treated with SDS by mixing 8.33 μM of each staple strand, 1% SDS and 63 mM Tris pH 7.5 in a total volume of 60 μl. The mixture was heated to 95�C for 2 minutes. Then scaffold, TAE buffer and MgAc was added so the final concentrations were 0.6 μM scaffold, 1X TAE, 12.5 mM MgAc with a total volume of 70 μl. Staple strand composition of the different structures are shown in Table 2.1.
Annealing
A PCR machine (VWR Collection DOPPIO Thermal Cycler) was used to perform the annealing. The program was 95�C for 5 minutes, a ramp of 3�C/s to 80�C,
