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Click Reaction Protocol

This protocols may be used as a starting point for optimization of your particular click chemistry procedures.
 
Preparation of the click Solution
The click solution (0.1 M CuBr / 0.1M 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 0.1M 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.

Considerations
Please read these considerations carefully before beginning your experiments to avoid typical problems that may arise when performing a click reaction.

The labeling 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 higher density labeling 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.

Please note: For this protocol, it is assumed that the azide is dissolved in DMSO. If the azide as well as the DNA is dissolved in an aqueous solution, TBTA - which is insoluble in water - can precipitate. In this case, a water-soluble ligand such as THPTA should be preferred when using the TCEP procedure. If the CuBr-method is being used or the use of THPTA is not possible and you see white precipitation clouds of TBTA, DMSO can be slowly added in small amounts to the solution. The solution should then be warmed to 30-40°C to solubilize the TBTA-ligand again. Please proceed very slowly and with small amounts of DMSO added gradually. If the solution is diluted with larger amounts of DMSO the reaction-efficiency can be reduced.

Please also note that the ligand and the copper-source should optimally be in the same solvent e.g. both in DMSO.

Important: EDTA as a buffer component should be avoided for low concentrations used. EDTA is a Cu(II)-complexing agent and thus “catches” a certain amount of catalyst from the solution. This can have a negative influence on the overall reactivity of the click reaction. This should be especially considered if EDTA was used and the yield was then lower than expected.
Click Procedure for short DNA oligos


Procedure using CuBr:
Ligand of choice: TBTA in a DMSO-containing solvent-system.
To 5 µl of a 2 mM DNA solution (10 nmol) in water, 5 µl of an azide solution (in DMSO, 10 mM, 50 nmol, 5 eq ), 3 µl of a freshly prepared solution containing 0.1 M  CuBr and 0.1M 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 3-4 h. The reaction is subsequently diluted with 0.3 M NaOAc (100 µl) and the DNA precipitated using 1 ml cold EtOH.
The washed residue is redissolved in pure water (20 µl) and can be used without further purification.
 

Click procedure using alternative Cu (I) sources

Procedure using TCEP:
Ligand of choice: THPTA (TBTA is also possible, but may precipitate in aqueous solutions)
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 THPTA 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 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. Instead of TCEP, also sodium-ascorbate can be used.

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 (8 mM, diluted with 10 mM Tris with 5 % T-BuOH from a stock of 0.1 N in DMSO) and precomplexed Cu(I) was added   (10 mM, 1 mg  CuBr (99-99%) dissolved in 700  µl of 10 mM THPTA 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.