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Protocol for Preparing DNA Conjugated Gold Nanoparticles
Deprotecting Thiolated Oligos:
Thiol modified and fluorophore conjugated oligonucleotides are purchased from IDT. They come in a “protected” state (thiol end is protected) and must be “deprotected” before they can be conjugated to AuNP.
1. Modified oligonucleotides are deprotected with 200 uL of 100 mM DTT in HEPES or TE buffer for 1.5 hours at room temperature
· 1.54 g DTT in 10 mL water will yield 1 M DTT
· Dilute to 100 mM in HEPES buffer or TE (180 uL TE with 20 uL 1 M DTT)
· Vortex, set on rocker for 60-90 minutes
· Bring up to 500 uL with NaPhos 5 mM
2. Purified using a NAP-5 column (GE Healthcare)
· Tape Nap5 column above a 50 ml conical
· Allow liquid to elute out
· Fill with 5 mM NaPhos and drain, repeat this 3x
· Add 500 ul of Oligos to the Nap5 column and allow liquid to drain through
· Move column over a series of Eppendorf tubes (labeled 1-10) and add 100 uL of NaPhos, allow liquid to drain into tube #1. Repeat for tubes 2-10.
· Spec oligo concentration by typing in the sequence into the NanoDrop. Combine the 4-5 tubes with the highest concentration.
· Freeze oligos until ready to attach to gold.
Materials for making DNA-AuNP:
· 470 uL Au (15 nM, Ted Pella)
· 10 uL NaPhos 0.5 M, pH 8
· 0.5 uL 10% SDS
· 20 uL Oligo-SH ~250 uM (deprotected)
Method:
1. Mix materials listed above and place on rocker for 20 minutes at room temperature
2. Add 6.25 uL 4M NaCl in 10 mM NaPhos pH 8, SDS 0.01%, vortex, sonicate for 10 seconds and set on rocker for 20 minutes are room temperature.
3. Repeat step #2
4. Add 13.5 uL 4M NaCl in 10 mM NaPhos pH 8, SDS 0.01%, vortex, sonicate for 10 seconds and set on rocker for 20 minutes are room temperature.
5. Repeat step #4 eight times
6. Leave on rocker overnight at room temperature
7. Spin for 30 minutes at room temperature at full speed
8. Remove supernatant, resuspend in 50-100 uL PBS, vortex. Sonicate if AuNP look like “flecks,” DNA-AuNP should look “oily” in PBS.
9. Repeat steps 7-8 two more times
10. Resuspend in a final volume of 50 uL (sonicate if necessary)
Annealing Complimentary Oligo:
1. Add the complementary oligo (conjugated to drug or flare) to 50 uL Au-oligo at 100-fold excess the concentration of AuNP. (75 uL max) Vortex, sonicate, quick spin down.
2. Heat to 80° C then slow cool using the “SLOW COOL” protocol in the thermal cycler
3. Transfer to ice bucket and keep on ice/at 4° C indefinitely
4. Wash with ice cold PBS and spin at full speed for 30 minutes to pellet spin at 4° C (in cold room) Repeat step 3 times.
5. Re-suspend in ~25-50 uL PBS, store at 4° C
Measure concentration of Gold Nanoparticles:
1. Spec on NanoDrop using UV Vis option at absorbance of 520 nm
2. Use Beer’s Law
3. Calculate “concentration”
4. Decadic Absorbance: value obtained by NanoDrop
5. Molar Absorptivity: 2.4*10^8
6. Path Length: 0.1 cm
7. Example:
8. Adjust volume to obtain desired concentration
Measure concentration of oligos conjugated to AuNP:
1. Treat 5 uL, 10 uL and 15 uL of DNA-AuNP (of a known AuNP concentration) overnight in 100 uL of 0.5M DDT and 200 mM NaPhos pH 8 (separates
2. Spin at room temperature at full speed and collect supernatant (supernatant should contain DNA, Au should be pelleted at the bottom)
3. Resuspend in 50 uL PBS and use 50 uL OliGreen reagent
4. Compare to standard curve generated from Quant-iT OliGreen
5. Product information and Manual:
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