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

(Difference between revisions)
 Revision as of 09:19, 24 September 2013 (view source) (→Calculating [AuNP] in Au@SiO2/DNA-MB solutions)← Previous diff Current revision (09:34, 25 September 2013) (view source) (→Notes) (8 intermediate revisions not shown.) Line 34: Line 34: x = 6.25nM 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 + + + {| class="wikitable" + |- + ! 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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## Calculating [AuNP] in Au@SiO2/DNA-MB solutions

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

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

x = 4.975nM

(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

(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.