User:Allison K. Alix/Notebook/Thesis Research/2013/05/31

Sample Prep

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Objectives Prepare an AuNP/DNA sample Prepare ThT/DNA sample Prepare MB sample Prepare ThT sample Prepare 0.05M phosphate buffer Procedures Part 1: Preparation of AuNP-DNA sample Final concentrations of AuNP and DNA should be 14.835nM and 1.11625μM respectively 1) 53.1μM(x)=(4.4506μM)(250μL) x= 20.95μL in 229.05μL PBS mix 20.95μL 53.1μM thiol-DNa with 229.05μL PBS 2) Mix 250μL DNA solution with 250μL 100mM DTT in 4% TEA NEW [DNA] = 2.225μM 3) React 10 minutes 5) Extract DTT from solution using 4 2mL aliquots of ethyl acetate 6) Mix 232.4μL DNA, 232.4μL 29.7nM AuNP, and 36.2μL sodium citrate buffer NEW [DNA] = 1.034μM NEW {[AuNP] = 13.805nM 7) React 10 minutes 8) Centrifuge for 7 minutes at 10000rpm Part 2: Preparation of thiol-DNA/ThT solution concentration of DNA must match [DNA] in AuNP/DNA solution and [ThT] must be at least twice this 1) 5.82μM(x)=(2.2253μM)(250μL) x = 95.6μL in 154.4μL PBS to make 250μL 2.2263μM DNA 2) 120μM(x)=(250μL)(4.44μM) x = 9.25μL in 240.75μL PBS to make 250μL 4.44μM ThT 3) Mix 250μL 2.2263μM DNA with 250μL 4.44μM ThT NEW [DNA] = 1.11265μM NEW [ThT] = 2.22μM Part 3: Preparation of MB 1) 7.61μM(x)=(100μL)(1.268μM) x = 16.7μL 7.61μM MB in 83.3μL PBS to make 100μL 1.268μM MB Part 4: Preparation of ThT concentration of ThT must match [ThT] in DNA/ThT sample 1) 120μM(x)=(250μL)(2.22μM) x = 4.625μL in 245.375μL PBS to make 250μL 2.22μM ThT Part 5: Preparation of 0.05M phosphate buffer, pH=7.4 *to be used to attach DNA to silica coated AuNP [Base] = potassium phosphate, monobasic = 136.09 g/mol [Acid] = potassium phosphate, dibasic = 174.20 g/mol pH = pKa + log(Base)/(Acid) 7.4 - 6.8 = log(Base)/(Acid) Base/Acid = 3.98 Acid + Base = 0.05M Base= 0.05M-Acid (0.05M-Acid)/Acid = 3.98 [Acid] = 0.0100M = 1.742g [Base] = 0.0400M = 5.4436g NOTE that this is based on 1L of buffer being used and only 500mL was actually prepared. One half of the masses above were added to ~400mL of water. The pH was adjusted to 7.4 using ~2M NaOH and the remaining water was then added.