User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/10: Difference between revisions
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====Procedure==== | ====Procedure==== | ||
*Combine 150μL AuNPs@SiO<sub>2</sub>, 15μL APS and 235μL ethanol | *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) | *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.> | Volumes may be doubled to avoid waste of SSMCC by using only 1 of 2mg.<br.> | ||
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Revision as of 13:19, 20 June 2013
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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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