Purification of CFET Tags

Excess dye NHS ester is removed by the size exclusion chromatography column followed by gel electrophoresis to eliminate the unlabeled and partially dye-labeled oligonucleotides. The CFET tags are desalted before use.

Fig. 2. A synthetic scheme for the preparation of a combinatorial fluorescence energy transfer tag consisting of FAM, TAMRA, and Cy5.

3.2.1. Removal of Excess Dye NHS Ester

1. Add sufficient volume of 0.1 M TEAA to the dye conjugation reaction products such that the total volume is 100 ||L. Vortex the solution.

2. Remove the top cap of the PD-10 column and pour off the excess liquid.

3. Cut the bottom tip of the column and support it over a suitable receptacle.

4. Equilibrate the column bed with 25 mL of 0.1 M TEAA.

5. After the buffer has run into the column, add the 100-|L reaction products to the central portion of the column's exposing surface followed by adding a few drops of 0.1 M TEAA.

6. When all the products have just entered the column bed, add 2.5 mL of 0.1 M TEAA.

7. Add 1.0 mL of 0.1 M TEAA and collect the eluate that contains the dye-labled oligonucleotide (see Note 5).

8. Lyophilize the eluate in a vacuum centrifuge.

3.2.2. Removal of Nondye-Labeled and Partially Dye-Labeled Oligonucleotides

1. Prepare the gel solution for electrophoresis by mixing 72 g urea, 90 mL 19:1 (v/v) 40% acrylamide (w/v)/N,W-methyl-fo"s-acrylamide, 16 mL 10X TBE buffer, and 45 |L water.

2. Stir the gel solution until a clear solution is obtained.

3. Degas the gel solution for 5 min.

4. Add 320 mL of 10% (w/v) fresh ammonium persulfate solution followed by vigorous mixing (approx 2 min).

5. Add 40 |L of TEMED with gentle mixing for 30 s.

6. Introduce the gel mixture immediately between the precasted glass plates (40 cm long) and insert a comb (0.8 mm thick) (see Note 6).

7. Prepare a sufficient quantity of 1X TBE buffer to fill both anodal and cathodal chambers by diluting a 10X TBE stock.

8. Mount the glass plates onto the electrophoresis apparatus.

9. Pre-electrophorese the gel at 65 W (1800 V, 40 mA) for approx 30 min.

10. Dissolve each lyophilized unpurified CFET tag in 12 ||L 95:5 (v/v) formamide/ 10 mM EDTA. Vortex to completely dissolve the sample.

11. Flush the wells in the gel with buffer from the upper reservoir and carefully load the samples.

12. Electrophorese at 65 W (1800 V, 40 mA) for at least 3 h. Cover the glass plates with an aluminum foil to avoid the CFET tags from exposing to light.

13. Separate the glass plates and carefully cut out the product bands (color from the CFET tags can be seen by naked eye) to prevent contamination by nearby impurities (see Note 7).

14. Elute the CFET tags in 1 mL of 0.1 M TEAA, pH 7.0, overnight at 4°C.

3.2.3. Desalting of CFET Tags

1. Wash the OPC with 5 mL ACN and 5 mL of 2.0 M TEAA.

2. Pass the samples through the OPC at a rate of about one drop per second and collect the eluate.

3. Pass the eluate through the OPC a second time.

4. Pass 15 mL of 0.1 M TEAA through the OPC.

5. Elute the samples from the OPC by 1 mL of 50% (v/v) ACN.

6. Determine the CFET tag concentration and store any unused material as a lyo-philized solid or in neutral aqueous media at -20°C.

3.3. Purification of Biotinylated Oligonucleotides

1. Dilute the deprotected oligonucleotide with three parts water.

2. Wash the OPC with 5 mL of ACN followed by 5 mL of 2.0 M TEAA.

3. Pass the oligonucleotide through the OPC at a rate of about one drop per second and collect the eluate.

4. Pass the eluate through the OPC a second time.

5. Pass 5 mL of Solution A through the OPC (see Note 8).

6. Use a new syringe and pass 10 mL of water through the OPC.

7. Apply a new syringe to the OPC. Gently push 1 mL of 3% TFA (v/v) through the OPC and incubate the oligonucleotide for 5 min to ensure complete detritylation. Then gently flush the remaining amount of TFA (4 mL) through the OPC.

8. Apply another new syringe to the OPC and pass through 10 mL of water.

9. Elute the oligonucleotide from the OPC with 1 mL of solution B.

10. Add 0.5 mL of concentrated NH4OH to the eluted oligonucleotide in solution B. Leave the mixture at room temperature for 15 min to achieve complete elimination of the side chain to produce a 3'-biotinylated oligonucleotide with a 5'-phos-phate.

11. Determine the oligonucleotide concentration and store any unused oligonucleotide as a lyophilized solid or in neutral aqueous media at -20°C.

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