🧬  NHS ester labeling of amino moeities

This protocol explains how to conjugate amino-labeled oligonucleotides with NHS.

🚛 Reagents needed 📦 :
Item Item article number Distributor Vendor
Dimethyl sulfoxide, anhydrous, ≥99.9% 276855-100ML
Merck SigmaAldrich
Sodium carbonate, anhydrous
223484 Merck SigmaAldrich
Sodium bicarbonate
S5761 Merck SigmaAldrich
⚖️ Scale of synthesis:

How much of the oligo do you wish to conjugate?     nmol

At what concentration is your stock oligo?  µM

What is the molecular weight of your oligo?*  g/mol

*if you don't know the molecular weight but have the DNA sequence use the Analyze Oligonucleotide calculator. Add XX g/mol for the mw of amino-C6.

1️⃣  If you don't have a stock of 0.5 M Na2CO3/NaHCO3 buffer (pH 8.75) prepare this.
The carbonate/bicarbonate anion system has two pKa values, one at 6.4 and one at 10.3. Therefore, at pH 8-9, you will have both carbonate and bicarbonate present. One simple way to make your buffer is to make two 100mM solutions, one of NaHCO3 and one of Na2CO3. You can then mix various proportions of the two until you get the proper pH.

 Make 0.5 M carbonate solution by adding 5.5 g in 100 mL nuclease-free water.

 Make 0.5 M bicorbonate solution by adding 4.2 g in 100 mL nuclease-free water.

 Combine 4 mL of carbonate solution from  and 46 mL of bicarbonate solution.

 Check pH and adjust.

 Fill up to 200 mL with nuclease-free water.

2️⃣  Oligos used for NHS conjugation should be dissolved in 0.5 M Na2CO3/NaHCO3 buffer (pH 8.75) as a stock.
      Preferably at 1 mM stock concentration.
      Measure the oligo on a NanoDrop for reference so that final yield can be calculated later.

3️⃣  Dissolve NHS ester in DMSO.

As a rule of thumb for  high reaction yield you'll need the NHS ester in 8 times molar excess to the oligonucleotide:

    NHS ester weight [mg]   = 8 ×     oligo weight [mg]         ×    NHS ester molar weight [Da]           / Oligo molar_weight [Da].
 = 8 ×  ×  / 

If you are dissolving for a stock solution perform all work under flow of inert gas and store the stock in a desiccator in -20°C.

Dissolve NHS ester using DMSO in 1/10 of the final reaction volume.

4️⃣  Dissolve the amino-oligo in 9/10 of the reaction volume using 0.5 M Na2CO3/NaHCO3.

5️⃣  Add the diluted amino-oligo into the DMSO dissolved NHS ester. You might see white precipitate at this point.

Reagent Volume Amount Final concentration
NHS ester dissolved in DMSO 10 µl X nmol µM
Amino-oligonucleotide in Na2CO3/NaHCO3 (pH 8.75) 90 µl 6 nmol µM
TOTAL: 100 µl

6️⃣  Vortex and wrap the Eppendorf tube in foil.
     ��� Incubate overnight 🌃 🛌  on a tube rotator.

🍚 Purification

Select purification method depending on required purity as well as the length of the oligonucleotide.
 If the oligonucleotide is ≥ 16 nt then use the standard Oligo Clean & Concentrator spin column from Zymo.
 If oligo length is ≥ 8 nt but <16 nt then use PAGE purification followed by Amicon filter cleanup.

 🐢 PAGE purification

 Set up the TBE-Urea gel (10% or 15%) to run in 1x TBE buffer. Use a syringe to clean each well.

 Run the gel for 1h at 300 Volt before loading the gel. This helps even the temperature in the gel and help denaturing. 

 Load a maximum of ~1 µg per well (for 9mer ssDNA mw ~2718.6 g/mole).
    The molecular weight, mw, of an oligo can roughly be approximated by its length, ntmw = 308.95×nt - 62

Reagent Amount Final concentration
Sample (16'311.6 ng) 30 µl 163.116 ng/µl
Formamide 70 µl 70%
Total: 100 µl

 Load 2-10 µl per well and run the gel until bands are clearly distinguishable to be cut out.

 Use a razor blade to cut out the upper 1/2 to 2/3 of the band of interest.

⛏️💥 💦 'Crush-and-Soak' recovering 🎣 of oligonucleotides from Polyacrylamide Gel 🍮

 Transfer the gel slice to a microcentrifuge tube. Use a sterile glass rod to crush the slice against the wall of the tube.

 Add 500 µl of 0.1 M sodium acetate (pH 6.0).

 Incubate at 90°C for 5 min.

 Push the tube down onto dry ice (-70°C), or into a -80°C freezer. Incubate for 5 min.

 Thaw and transfer to an Amicon Ultra-0.5 Centrifugal Filter Unit (3 kDa cut-off).

 Spin for 30 min at 14,000 x g.

🐇 Oligo clean & concentrator spin column
✋ Notes 🛑❗

If it is the first time you use the kit add 96 ml 100% ethanol (104 ml 95% ethanol) to the 24 ml DNA Wash Buffer concentrate (D4060)
or 192 ml 100% ethanol (208 ml of 95% ethanol) to the 48 ml DNA Wash Buffer concentrate (D4061).

 If the reaction is 30 µl add 20 µl of nuclease-free water to the 30 µl reaction mixture for a total of 50 µl.

 Add 100 µl Oligo Binding Buffer to 50 µl sample.

 Add 400 µl ethanol (95-100%) and mix well by pipetting.

 Transfer the 450 µl sample to the Zymo spin column.

 Add 750 µl DNA Wash Buffer to the column and centrifuge for 1 min at 10,000-16,000 x g.

 Transfer the column to a new Eppendorf tube.

 Add ≥ 6 µl elution buffer (water or TE) to the center of the column.

 Centrifuge for 1 min at 10,000-16,000 x g.

The flow-through can immediately be used or stored frozen.
Measure the yield on NanoDrop and then elute further to desired stock concentration (usually 100 µM).