Biomod/2014/fit Method.html: Difference between revisions
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<h3><p> | <h3><p>・Artificial experiment using fluorescent molecule<br> | ||
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<td><div style="width:210px;"><img alt="" src="/images//5/56/Fitfisuku.jpg" width="200" height="150" border="0"/ align="left" style="margin-right: 10px;"><p>(*^_^*)</p></div></td> | <td><div style="width:210px;"><img alt="" src="/images//5/56/Fitfisuku.jpg" width="200" height="150" border="0"/ align="left" style="margin-right: 10px;"><p>(*^_^*)</p></div></td> | ||
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| Actually, when observe it, tested it using the material which is similar that I adorned it to DNA to confirm it how much the density of fluorescence molecules can measure if there is it. The fluorescence molecules which it modified to DNA is FITC and TAMURA、Fluorescein in substitution for FITC, used Rhodamin B in substitution for TAMURA in artificial experiment.<br clear="all"></td></table> | ||
</p><br> | </p><br> | ||
<p> | <p>・Synthesis of meso porous silica particle<br> | ||
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| N-cetyltrimethylammonium bromide (CTAB, 1.0009 g) was first dissolved in 50 ml of pure water by heating. After cooling to room temperature, aqueous ammonia (13 ml) and ethanol (75 ml) were added. The mixture was stirred for 15 min and TEOS (1.94 ml) added rapidly while stirring was continued. TEOS (30 μl) and APTES (30 μl) were introduced later. The mixture was allowed to stir for 2 h to give rise to white precipitates. The solid product was filtered, washed with deionized water and methanol, and dried in air. meso porous silica is synthesized Particle and pour site was determined by SEM observation.To remove the surfactant template (CTAB), the white powder was refluxed for 16 h in a solution of 1.00 ml of HCl (37%) and 80.00 ml of methanol followed by extensively washing with deionized water and methanol. Afterwards when we absorb it and filter it. After absorbing it, having filtered it, we remove water by putting meso porous silica in the vacuum state. The resulting surfactant-removed amine-functionalized MSN (MSN-NH2) was placed under high vacuum to remove the remaining solvent in the mesopores. The MSN-NH2 (50 mg) was reacted with succinic anhydride (1.00 g) in N,N-dimetylformamide solution (20 ml) under N2 gas for 8 h with continuous stirring.After a thorough water wash, the carboxylated nanoparticles (MSN-COOH) were activated using EDC (0.1471g) and sulfo-NHS (0.132g) in a MES buffer (pH 6.0) for 15 min at room temperature with continuous stirring. Twenty microliters of PBS buffer (100mM, pH7.4) was then added in the mixture, followed by the addition of TAMRA labeled DNA (5μl 100μM) at room temperature with continuous stirring for 6 h and washing in PBS buffer (0.1 M,pH 7.4) to form the resultant DNA-conjugated nanoparticles (MSN-DNA or -cDNA). | ||
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Revision as of 11:26, 29 August 2014
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<title>........</title> </head> <body style="background-color:#EOFFFF;"> <div id="pagebody">
<div id="header"><h1><p style="background:#CCFFFF; color:purple;"><font face=cursive size="6"><B> Team FIT </font> </B></p></a></h1></div>
<table> <tr align="center"> <td cellspacing="10" cellpadding="10" width="1055" width="180" height="60" bgcolor="#BAD3FF"><a href="http://openwetware.org/wiki/Biomod/2014/Fukuoka"><B><font face=cursive color="#003366" size="3">Top</font></B></a></td> <td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Introduction.html"><B><font face=cursive color="#003366" size="3">Introduction</font></B></a></td> <td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Approach and Goals.html"><B><font face=cursive color="#003366" size="3" >Approach and Goals</font></B></a></td> <td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Method.html"><B><font face=cursive color="#FF6633" size="3">Method</font></B></a></td> <td cellspacing="10" cellpadding="10" width="1300" width="180" bgcolor="#BAD3FF"><a href="fit_Results and Discussion.html"><B><font face=cursive color="#003366" size="3">Results and Discussion</font></B></a></td> <td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Member.html"><B><font face=cursive color="#003366" size="3">Member</font></B></a></td> <td cellspacing="10" cellpadding="10" width="1055" width="180" bgcolor="#BAD3FF"><a href="fit_Sponsor.html"><B><font face=cursive color="#003366" size="3">Sponsor</font></B></a></td> </tr></table>
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<h2 p style="background:#FFFFFF; color:#003366;"><font face=cursive size="5"><B> Method </font></B></p></h2>
<div style="padding: 35px;">
<h3><p>・Artificial experiment using fluorescent molecule<br> <table> <td><div style="width:210px;"><img alt="" src="/images//5/56/Fitfisuku.jpg" width="200" height="150" border="0"/ align="left" style="margin-right: 10px;"><p>(*^_^*)</p></div></td>
<td>
Actually, when observe it, tested it using the material which is similar that I adorned it to DNA to confirm it how much the density of fluorescence molecules can measure if there is it. The fluorescence molecules which it modified to DNA is FITC and TAMURA、Fluorescein in substitution for FITC, used Rhodamin B in substitution for TAMURA in artificial experiment.<br clear="all"></td></table>
</p><br>
<p>・Synthesis of meso porous silica particle<br>
<table>
<td><div style="width:210px;"><img alt="" src="/images//7/71/Fitsilicanh.jpg" width="200" height="150" border="0"/ align="left" style="margin-right: 10px;"><p>(*^_^*)</p></div></td>
<td>
N-cetyltrimethylammonium bromide (CTAB, 1.0009 g) was first dissolved in 50 ml of pure water by heating. After cooling to room temperature, aqueous ammonia (13 ml) and ethanol (75 ml) were added. The mixture was stirred for 15 min and TEOS (1.94 ml) added rapidly while stirring was continued. TEOS (30 μl) and APTES (30 μl) were introduced later. The mixture was allowed to stir for 2 h to give rise to white precipitates. The solid product was filtered, washed with deionized water and methanol, and dried in air. meso porous silica is synthesized Particle and pour site was determined by SEM observation.To remove the surfactant template (CTAB), the white powder was refluxed for 16 h in a solution of 1.00 ml of HCl (37%) and 80.00 ml of methanol followed by extensively washing with deionized water and methanol. Afterwards when we absorb it and filter it. After absorbing it, having filtered it, we remove water by putting meso porous silica in the vacuum state. The resulting surfactant-removed amine-functionalized MSN (MSN-NH2) was placed under high vacuum to remove the remaining solvent in the mesopores. The MSN-NH2 (50 mg) was reacted with succinic anhydride (1.00 g) in N,N-dimetylformamide solution (20 ml) under N2 gas for 8 h with continuous stirring.After a thorough water wash, the carboxylated nanoparticles (MSN-COOH) were activated using EDC (0.1471g) and sulfo-NHS (0.132g) in a MES buffer (pH 6.0) for 15 min at room temperature with continuous stirring. Twenty microliters of PBS buffer (100mM, pH7.4) was then added in the mixture, followed by the addition of TAMRA labeled DNA (5μl 100μM) at room temperature with continuous stirring for 6 h and washing in PBS buffer (0.1 M,pH 7.4) to form the resultant DNA-conjugated nanoparticles (MSN-DNA or -cDNA).
<br> <br clear="all"></td></table> </p> <p>・キャラクタ<br> <table> <td><div style="width:235px;"> <img alt="" src="/images//c/cd/Fitokayama.jpg" width="225" height="300" border="0"/ align="left" style="margin-right: 10px;"><p>(*^_^*)</p></div></td>
<td>
二種類の蛍光分子による相互作用を確認するため予備実験を行った。単一系と複合系それぞれの励起,蛍光波長を分光光度計を用いて測定した。<br> メソポーラスシリカはSEMを用いて観察し、粒子の大きさや孔の観察を行った。<br> DNAを修飾したシリカの観察は共焦点顕微鏡を用いて観察した。本来ならば、蛍光顕微鏡を用いた方がよりきれいなデータをとることができるが液量がとても少なかったため共焦点顕微鏡を用いて観察した。</td><br></table></p>
</p></h3>
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