User:Allison K. Alix/Notebook/Thesis Research/2013/09/24

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(Preparation of Au/DNA/MB w/o Si for FCS)
Current revision (10:34, 25 September 2013) (view source)
(Notes)
 
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2) Extract DTT with 4 2mL aliquots of ethyl acetate
2) Extract DTT with 4 2mL aliquots of ethyl acetate
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3) Prepare 100uL 12.5nM AuNP
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23.5(x) = 12.5(100uL)
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x = 53.2uL in 46.8uL water
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4) mix 20uL DNA with 20uL AuNP and 5uL 268mM sodium citrate. React 10 minutes.
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5) Centrifuge 7 minutes at 10000rpm.
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6) Add 6uL MB heat for 25 min at 70 C
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==Notes==
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FCS Measurements for the Au@Si/DNA-MB samples were run twice each for 7 minutes at a time. Looking at the N values, we have determined that the range for detection is between the 50X and 100X diluted samples (AuNP concentrations of 110pM and 55.2pM respectively.) The Au/DNA-MB samples were not run for this day as they required a few hours to cool to room temperature. This being said, they will also be run in this same 50X-100X diltuion range.

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Calculating [AuNP] in Au@SiO2/DNA-MB solutions

  • Original [Au@SiO2] = 5nM before attaching DNA

1) Addition of 5uL APS

(1mL)(5nM)=(1.005mL)(x)

x = 4.975nM

2) Addition of 1.5mL Sulfo-SMCC

(1.005mL)(4.975nM) = (x)(2.505mL)

x = 1.996nM

3) Centrifugation, removal of supernatant, and redispersion in 530uL KP buffer

(1.996nM)(2.505mL) = x(0.530mL)

x = 9.43nM

4) Addition of 270uL DNA

(9.43nM)(530uL)=x(800uL)

x = 6.25nM

5) Addition of 13.3uL 7.61uM MB to 100uL Au@SiO2/DNA

6.25nM(100uL)=x(113.3uL)

x = 5.52nM


Dilution Factor [Au@SiO2] [DNA] [MB]
None (stock) 5.52nM 802nM 895nM
2X 2.76nM 401nM 447.5nM
10X 552pM 80.2nM 89.5nM
100X 55.2pM 8.02nM 8.95nM
1000X 5.52pM 802pM 895pM
10000X 552fM 80.2pM 89.5pM

Preparation of Au/DNA/MB w/o Si for FCS

Final Concentrations need to be

[Au] = 6.25nM

[DNA] = 910nM

1) Mix 145.9uL 3.64uM thiol-DNA with 145.9uL 100mM DTT in 4% TEA. React 10 minutes

53.1uM(10uL) = 3.64uM(x)

10uL in 135.9 water

2) Extract DTT with 4 2mL aliquots of ethyl acetate

3) Prepare 100uL 12.5nM AuNP

23.5(x) = 12.5(100uL)

x = 53.2uL in 46.8uL water

4) mix 20uL DNA with 20uL AuNP and 5uL 268mM sodium citrate. React 10 minutes.

5) Centrifuge 7 minutes at 10000rpm.

6) Add 6uL MB heat for 25 min at 70 C

Notes

FCS Measurements for the Au@Si/DNA-MB samples were run twice each for 7 minutes at a time. Looking at the N values, we have determined that the range for detection is between the 50X and 100X diluted samples (AuNP concentrations of 110pM and 55.2pM respectively.) The Au/DNA-MB samples were not run for this day as they required a few hours to cool to room temperature. This being said, they will also be run in this same 50X-100X diltuion range.





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