User:Allison K. Alix/Notebook/Thesis Research/2013/08/12

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To Do

  • AuNP@SiO2/DNA attachment procedure (10X diluted AuNP@SiO2 prepared with 2uL TES to be used)

AuNP@SiO2/DNA attachment

adapted from Madeleine Y. Bee[1]

1) Prepare a fresh 10X dilution of AuNP@SiO2

  • Sonicate AuNP@SiO2 for 10 minutes
  • Mix 15uL AuNP@SiO2 with 135uL filter milliQ water (@ room temperature), vortex to mix thorooughly

2) Mix 150uL AuNP@SiO2 with 15uL APS and 235uL 100% ethanol. Sonicate for 30 minutes

3) Centrifuge and rinse solution a total of 5 times at 9100rpm for 5 minutes. Redisperse in ethanol for the first three and 10mM sodium phosphate buffer (pH=9) for the last two.

NOTE: after first round of centrifugation, it was apparent 5 minutes was a sufficient amount of time. However, after removal of the supernatant and redispersion in ethanol, there was difficulty getting the particles back into solution. They were allowed to sonicate for 10 minutes to try to induce separation but still looked relatively aggregated. To further induce separate, the sample was subjected to ultrasonic liquid processing for ~15 seconds, which proved to be effective. The sample was only rinsed with phosphate buffer once, as it would not separate even after long periods of centrifugation Change centrifugation time to 30 seconds.

4) Dilute 2mg Sulfo-SSMC with 200uL sodium phosphate buffer. Transfer to a larger epi-test tube and continue to dilute until total volume reaches 800uL. Vortex to completely dissolve.

5) Mix 400uL Sulfo-SSMC with 400uL AuNP@SiO2/NH2 solution. Sonicate 1 hour.

6) While solution is sonicating. Reduce S-DNA to HS-DNA. Mix 50uL 40uM S-DNA with 50uL DTT in TEA solution. React 10 minutes and extract DTT using triethylacetate.