1. Pour the synthesized solid support to an Eppendorf tube (with or without screw cap). Start from half of the support, in case any failure experiment happens.

 

2. Add 1 mL conc. Ammonium hydroxide strong base to the tube (Ammonium hydroxide is aliquoted and stored in refrigerator to keep fresh). Make sure the support is submerged in the solution. Tightly seal the tube with parafilm, or use the cap with screw to seal it. Heat the sample at 55oC for 16h. Cool down the sample to room temperature. The DNA is now cleaved from the support and fully deprotected.

 

3. Transfer the supernatant to a new Eppendorf tube. Wash the support with 0.4 mL clean water twice, and combine all the supernatants from every washing.

 

4. If the Glen-pak DNA cartridge is used for the DMTr-on purification, Ammonium hydroxide is not needed to be evaporated. Go to step 5a. If HPLC is used for the DMTr-on purification, Ammonium hydroxide is needed to be evaporated to get a neutral solution. Use Speed-vac at 4 oC to evaporate Ammonium hydroxide for ~1h. Don’t heat the sample during evaporation since it may cause de-tritylation. Go to step 5b.

 

DMTr-on purification

5a. Follow the Glen Research Purification of DNA Oligonucleotides (DMT-ON) protocol. https://www.glenresearch.com/media/productattach/import/tbn/GlenPak_UserGuide.pdf

The de-tritylation and washing off failure oligos will happen on the cartridge. The DMTr-off DNA is in solution of MeCN/water mixture. Use speed-vac to evaporate MeCN, for the second purification.

 

5b. Use Waters preparative HPLC system. Buffer A is water, buffer B is 50% MeCN in water, both containing 20mM TEAB pH 7.6. Filter the DNA sample before injection. Collect the full length DMTr-on DNA product (typically washed out at 60% buffer B), lyophilize the sample to dryness. Re-dissolve the DNA in 0.2 mL water, and transfer to a new Eppendorf tube. Wash the tube with 0.2 mL water twice and combine them to maximize the recovery yield. Add 3% trichloroacetic acid in water to the DNA solution for the de-tritylation. Typically 50-100 μl of acid is enough. Monitor the pH of the solution if necessary. pH ~1 is the acidity for de-tritylation to happen, and sometimes white precipitation can be observed. Incubate the sample at room temperature for 5 min and DMTr group should be cleaved off. Add ~100 μl 2M TEAAc or TEAB buffer, pH 7, to the solution to neutralize the TCA acid. Monitor the pH of the solution if necessary. Filter the sample by spin column for second HPLC purification.

 

DMTr-off purification

6a. Use Waters preparative HPLC system for DMTr-off purification. DMTr-off DNA is typically washed out at 30% buffer B. Collect the DNA, lyophilize the sample to dryness and re-dissolve in water. Quantify the DNA by UV, and prepare the sample with certain concentrations for the further experiment.

 

6b. Use PAGE gel to do the second purification. Follow our website online protocol for PAGE gel electrophoresis experiment, and recover the DNA from the gel. Remove the unnecessary salt by ethanol precipitation or Glen-pak desalting cartridge/DNA cartridge. Re-dissolve the DNA in water, quantify the sample and prepare for the further experiment.