Protocol Aptamer treatment with TCEP

3 x 12.1 nmoli APT = 36.3 nmoli

Pt 1 flacon: (Eu zic sa facem numai un flacon o data – ca 242 ul sol 50 uM APT  6050 ul sol 2
uM 60 el… in plus nu stim daca nu afecteaza TCEP, ca noi nu il putem separa dupa de APT…)

12.1 nmoli APT ……………………………………….… 121 ul TRIS = 0.121 ml TRIS

 100000 nmoli = 100 umoli …………………… 1000 ml TRIS  CM 100 uM

 121 ul sol 100 uM APT in TRIS+ 121 ul sol 20 mM TCEP in TRIS  242 ul sol 50 uM APT  1 h, in
the DARK, room temp  soc termic (5 min, 93-95 C, 20 s, -20C)

MTCEP = 286.65

20 mM TCEP …. 20 x 286.65=5733 mg…………….. 1000 ml

X = 0.693 mg ………………. 0.121ml  aprox 1-2 mg TCEP

Bibliografie

Activation and Pretreatment of Thiolated DNA and Linker DNA Prior to use, thiol-modified (thiolated)
DNA was activated with TCEP. Tipically, 3’ and 5’-thiolated DNA was treated with fresh prepared 100
μM TCEP (TCEP: DNA=100:1) in Tris-HCl buffer (pH 8.5) for 1 h at room temperature to cleave the
disulfide bond. The de-protected oligonucleotides were purified using a NAP-5 column (GE
Healthcare) before adding to the GNR solution for DNA decoration. In order to improve reaction
rate, linker DNA containg aptamer sequences was pretreated by heating it to 90 °C for 5 min,
immediately cooling to 4 °C for 10 min, and keeping it at room temperature for 5 min before adding
it to the reaction solution.

(https://doi.org/10.1039/C6RA04439E)

Prepare 10 mM TCEP. ▲ CRITICAL STEP TCEP should be freshly prepared. 9| Pipette 9 µl of 1 mM

DNA1 into a microcentrifuge tube and 9 µl of 1 mM DNA2 into another one. 10| Add 1 µl of 500 mM
acetate buffer (pH 5.2) and 1.5 µl of 10 mM TCEP to each tube to activate the thiol-modified DNA.
Incubate the sample at room temperature for 1 h. 11| Transfer 3 ml of the already prepared gold
nanoparticles to each of the two NaOH-treated glass vials, and then add the TCEP-treated thiol DNA
with gentle shaking by hand.

(https://doi.org/10.1038/nprot.2006.38)

“the stock solution of PSA aptamer (20 μM) was reduced with a 10 mM TCEP solution for one h at
25°C to break disulfide bonds. Subsequently, the stock solution of PSA aptamer was diluted with a
ratio of 1:20 with 0.1 M PBS (pH= 7.4). Afterward, 10 μL of aptamer solution (1 μM) was dropped
onto the Au…”

TCEP Reduction Protocol Note: TCEP does not interfere with coupling and does not require removal
prior to immobilization 1. Resuspend and aliquot aptamer in TE buffer to 100 µM according to the
instruction on the Product Information Form. 2. Prepare a 1:1 v/v dilution of aptamer solution with
20 mM TCEP solution, for a final concentration of 50 µM aptamer and 10 mM TCEP. Incubate the
reaction at room temperature for 1 hour. Folding Instructions Prior to Immobilization (see BP:
Handling & Storage for a more detailed protocol) 1. Prior to use, dilute the reduced thiol-aptamer to
its working concentration in Folding Buffer. 2. Heat the aptamer solution to 85 — 95 ºC for 5
minutes. 3. Allow the solution to cool to room temperature (~15 minutes).

(Base pair technologies)