User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/10: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Silica-coating Reaction</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Silica-coating Reaction</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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*Silica-coating procedure from [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/05/24 05/24/2013] performed<br.> | *Silica-coating procedure from [http://openwetware.org/wiki/User:Madeleine_Y._Bee/Notebook/Single_Molecule_Fluorescence/2013/05/24 05/24/2013] performed<br.> | ||
**MHA attachment performed, silica-coating reaction to be performed tomorrow | **MHA attachment performed, silica-coating reaction to be performed tomorrow | ||
*Sodium phosphate buffer, pH 9, made | |||
===Buffer Preparation and Calculations=== | ===Buffer Preparation and Calculations=== | ||
Sodium phosphate buffer, pH 9, to be used for the modification of HS-DNA and AuNPs@SiO<sub>2</sub> procedure.<br.> | Sodium phosphate buffer, pH 9, to be used for the modification of HS-DNA and AuNPs@SiO<sub>2</sub> procedure.<br.> | ||
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Use 0.0094g NaH<sub>2</sub>PO<sub>4</sub> and 1.19188g Na<sub>2</sub>HPO<sub>4</sub> in 1L water.<br.> | Use 0.0094g NaH<sub>2</sub>PO<sub>4</sub> and 1.19188g Na<sub>2</sub>HPO<sub>4</sub> in 1L water.<br.> | ||
===New: Modifying Silica-Coated AuNPs with HS-DNA Procedure=== | ===New: Modifying Silica-Coated AuNPs with HS-DNA Procedure=== | ||
*Combine 150μL AuNPs@SiO<sub>2</sub>, 15μL APS and 235μL ethanol | adapted from [http://pubs.acs.org/doi/full/10.1021/la7019846 Silica-Coated, Au/Ag Striped Nanowires for Bioanalysis] | ||
* | ====Reactions==== | ||
*Rinse sample three times by centrifugation at 7700g with ethanol and two times with sodium phosphate buffer, pH 9 (SPB) | =====Fuctionalizing the AuNPs@SiO<sub>2</sub> with NH<sub>2</sub>===== | ||
AuNPs@SiO<sub>2</sub><sub>(aq*)</sub>+H<sub>2</sub>N(CH<sub>2</sub>)<sub>3</sub>Si(OCH<sub>3</sub>)<sub>3</sub><sub>(l)</sub>→AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub><br.> | |||
'*'denotes colloidal system | |||
=====Activating the NH<sub>2</sub> Functionalized AuNPs@SiO<sub>2</sub> with Sulfo-SMCC===== | |||
AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub>+C<sub>16</sub>H<sub>17</sub>N<sub>2</sub>O<sub>9</sub>SNa<sub>(aq**)</sub>→SSMCC-Activated AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub><br.> | |||
'*'denotes colloidal system<br.> | |||
'**"denotes dissolution in buffer, not water | |||
=====Attaching the Thiolated DNA===== | |||
SSMCC-Activated AuNPs@SiO<sub>2</sub>-NH<sub>2</sub><sub>(aq*)</sub>+HS-DNA<sub>(aq)</sub>→Crosslinked S-DNA and AuNPs@SiO<sub>2</sub>-NH<sub>(aq*)</sub><br.> | |||
'*'denotes colloidal system<br.> | |||
====Chemicals Needed and Safety==== | |||
*Ethanol | |||
*15μL APS ((3-aminopropyl)trimethoxysilane) | |||
*1mg SSMCC (sulfosuccinimidyl-4-N-maleimidomethyl cyclohexane-1-carboxylate) | |||
**Must be stored at -20°C | |||
**Moisture sensitive, store in desiccant | |||
**[http://www.piercenet.com/instructions/2160581.pdf Manufacturer Instructions] | |||
*Potassium phosphate buffer, pH 7.4 | |||
*Sodium phosphate buffer, pH 9 | |||
*150μL Silica-coated AuNPs | |||
*50μL of 20μM HS-DNA | |||
====Procedure==== | |||
*Combine 150μL AuNPs@SiO<sub>2</sub>, 15μL APS, and 235μL ethanol | |||
*Sonicate solution for 30 minutes | |||
*Rinse sample three times by centrifugation at 7700g (9081.99rpm) with ethanol and two times with sodium phosphate buffer, pH 9 (SPB) | |||
*Make a separate solution of 1 mg sulfo-SMCC in 400μL of SPB | *Make a separate solution of 1 mg sulfo-SMCC in 400μL of SPB | ||
*Add SSMCC solution to AuNP solution | *Add SSMCC solution to AuNP solution | ||
* | *Sonicate for 1 hour | ||
*Rinse twice with SPB | *Rinse by centrifugation at 7700g (9081.99rpm) twice with SPB | ||
*Rinse twice with potassium phosphate buffer, pH 7.4 (KPB) | *Rinse by centrifugation at 7700g (9081.99rpm) twice with potassium phosphate buffer, pH 7.4 (KPB) | ||
*Combine sample with 50μL 20μM HS-DNA | *Combine sample with 50μL 20μM HS-DNA | ||
* | *Sonicate for 1 hour | ||
*Rinse three times with KPB | *Rinse by centrifugation at 7700g (9081.99rpm) three times with KPB | ||
*Re-suspend in 150μL KPB | *Re-suspend in 150μL KPB | ||
Since times for centrifuging were not specified, 5 minute intervals will be done to determine appropriate length of time.<br.> | |||
Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.> | |||
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Latest revision as of 22:45, 26 September 2017
Silica-coating Reaction | Main project page Previous entry Next entry |
June 10, 2013
Buffer Preparation and CalculationsSodium phosphate buffer, pH 9, to be used for the modification of HS-DNA and AuNPs@SiO2 procedure.<br.> monosodium phosphate- MW: 119.98g/mol<br.> disodium phosphate- MW: 141.96g/mol, pKA: 6.82<br.> buffer- pH 9,concentration 10mM<br.> <br.> 9=6.82+log([base]/[acid])<br.> 151.36=(10-[base])/([acid])<br.> [acid](151.36+1)=10<br.> [acid]=0.066mM<br.> [base]=9.935mM<br.> <br.> Use 0.0094g NaH2PO4 and 1.19188g Na2HPO4 in 1L water.<br.> New: Modifying Silica-Coated AuNPs with HS-DNA Procedureadapted from Silica-Coated, Au/Ag Striped Nanowires for Bioanalysis ReactionsFuctionalizing the AuNPs@SiO2 with NH2AuNPs@SiO2(aq*)+H2N(CH2)3Si(OCH3)3(l)→AuNPs@SiO2-NH2(aq*)<br.> '*'denotes colloidal system Activating the NH2 Functionalized AuNPs@SiO2 with Sulfo-SMCCAuNPs@SiO2-NH2(aq*)+C16H17N2O9SNa(aq**)→SSMCC-Activated AuNPs@SiO2-NH2(aq*)<br.> '*'denotes colloidal system<br.> '**"denotes dissolution in buffer, not water Attaching the Thiolated DNASSMCC-Activated AuNPs@SiO2-NH2(aq*)+HS-DNA(aq)→Crosslinked S-DNA and AuNPs@SiO2-NH(aq*)<br.> '*'denotes colloidal system<br.> Chemicals Needed and Safety
Procedure
Since times for centrifuging were not specified, 5 minute intervals will be done to determine appropriate length of time.<br.> Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.>
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