QIAquick Nucleotide Removal Kit (50)

Protocol tips

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	Follow manufacturer's instructions

Protocol tips
Follow manufacturer's instructions

Publication protocol

Adaptor-Fluorophore Conjugation
1. Resuspend the lyophilized oligonucleotide in 500 μl nuclease-free water.
• Note: The oligonucleotide given in the reagents/tools section reflects the sequence that we used in developing this procedure. For the Quick-irCLIP protocol, this exact sequence is not critical, and the sequence could be modified so long as the new oligonucleotide retained the 5’ phosphate, the internal azide, and the poly(A) stretch.
2. Using the 5’ DNA Adenylation Kit, prepare the adenylation reaction by adding 37.5 μL of 1 mM ATP, 37.5 μL 10x buffer, and 50 μL Methanobacterium thermoautotrophicum (Mth) RNA ligase to 250 μL resuspended oligonucleotide.
• Note: The remaining 250 μL resuspended oligonucleotide can be stored at −20 °C until needed for another adaptor preparation.
3. Incubate the reaction for 2 h at 65 °C.
4. Inactivate the reaction by incubating for 10 min at 85 °C.
5. Transfer the reaction (375 μL) to a new 1.5 mL microcentrifuge tube.
6. Add 40 μL 3 M sodium acetate (pH 5.5) and 1 mL 100% ethanol, mix well by vortexing, and precipitate overnight at −20 °C.
7. Pellet the precipitated, adenylated oligonucleotide by centrifugation at >16,000 RCF for 30 min.
8. Discard supernatant, being careful not to disturb the pellet, and wash with 500 μL ice-cold 80% ethanol.
9. Carefully remove ethanol and allow the pellet to air dry. Do not let the pellet over-dry as this may make resuspension difficult.
10. Resuspend each pellet in 180 μL 1x PBS.
11. Conjugate the infrared dye via “click” chemistry by adding 20 μL of 10 mM IRdye-800CW-DBCO to the 180 μL oligonucleotide solution and incubating for 2 h at 37 °C.
12. Column purify the final adaptor using the QIAquick Nucleotide Removal Kit.
a. To do this, mix the 200 μL “click” reaction with 4.8 mL PNI buffer and dispense 250 μL aliquots across 20 QIAquick nucleotide removal columns.
b. Follow the centrifugation and wash steps as described in the manufacturer’s protocol.
c. Elute using 50 μL nuclease-free water.
d. Pool fractions and quantitate using a Nanodrop (or similar). Concentration should be approximately 10 μM.
13. Aliquot infrared adaptor oligonucleotide and store at −20 °C in the dark

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Manufacturer protocol

Download the product protocol from Qiagen for QIAquick Nucleotide Removal Kit (50) below.

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