Preparation of the “Click Solution”

• The “click solution” (0.1 M CuBr / 0.1 M TBTA 1:2 in DMSO/t-BuOH 3:1) must always be freshly prepared prior to use!
• Dissolve 1 mg CuBr in 70 μl DMSO/t-BuOH 3:1 to obtain a 0.1 M solution. This solution must be freshly prepared and cannot be stored.
• Dissolve 54 mg TBTA in 1 ml DMSO/t-BuOH 3:1 for a 0.1 M solution. This solution can be stored at -20 °C.
• Add 1 volume of the 0.1 M CuBr solution quickly to 2 volumes of the 0.1 M TBTA solution to obtain “click solution”, ready to use.

Click Procedure for Short DNA Oligos

Procedure using CuBr: To 5 µl of a 2 mM DNA solution (10 nmol) in water,
2 µl of an azide solution (50 mM, 50 nmol, 5 eq.), 3 µl of a freshly prepared solution containing 0.1 M CuBr and 0.1 M TBTA ligand in a 1:2 ratio in 3:1 DMSO/t-BuOH is added. The mixture is thoroughly mixed and shaken at
25 °C for 3 h. The reaction is subsequently diluted with 0.3 M NaOAc (100 µl) and the DNA is precipitated using 1 ml cold EtOH. The supernatant is then removed and the residue is washed twice with 1 ml cold EtOH. The washed residue is re-dissolved in pure water (20 μl) and can be used without further purification.

Considerations for the CuBr method:

• The labelling reaction works more efficiently with concentrated solutions of alkynes (oligo) and azides (label).
  The best way to carry out the click reaction is to mix the oligo and the azide-label in a minimal amount of solvent.
• Alkyne / Azide ratio: from 1:2 to 1:10 for highdensity labelling reactions (e.g. 10 alkynes in a row).
• The click reaction is normally accelerated by elevated temperature and can be ready in less than 30 min when the reaction temperature is around 40 - 45 °C.
• The reaction time depends on: a) concentration of azide and oligo in the solution; b) reaction temperature; c) stirring and/or mixing of the solution.
• The work-up of the reaction is normally carried out by precipitation of the labeled oligo (addition of a salt solution, e.g. 0.3 M NaOAc        followed by addition of cold abs EtOH).

Click Procedure using alternative Cu(I) Sources

Procedure using TCEP: To 25 µl of a 0.5 mM DNA solution (12.5 nmol) in water, 6.25 µl of an azide solution (0.1 N, 625 nmol) and 10 µl of a solution containing Cu(II)-salt (CuSO4) and TBTA ligand in a 1:2 ratio in 4:3:1 water/DMSO/t-BuOH is added (0.05 N, 250 nmol). The mixture is vortexed and 5 µl of a freshly prepared tris-(2-carboxyethyl)-phosphine (TCEP) solution in water is added (0.1 N, 500 nmol). The solution is shaken at
15 °C over night and subsequently diluted with water (200 µl) and used for gel electrophoresis without further purification.

Click Procedure for a 300 bp PCR Product

To 10 µl DNA solution (1-4 pmol DNA, 10 mM Tris), 10 µl fluorescein azide solution (5 mM, diluted with 10 mM Tris with 5 % t-BuOH from a stock of
0.1 N in DMSO) and 10 µl pre-complexed Cu(I) was added (10 mM; 1 mg CuBr (99.99%) dissolved in 700 µl of 10 mM TBTA ligand in t-BuOH/DMSO 1:3) The sample is shaken at 37 °C for 2 h. Then formamide buffer is added and the samples are analyzed using a 5 % PAGE gel. Control experiments show that the reaction is completed in less than 30 min.

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