The method detailed in this protocol for the purification of crude synthesized oligonucleotides using denaturing polyacrylamide gel electrophoresis is a modification of the method used in Michael Smith's laboratory (University of British Columbia, Vancouver, British Columbia) for more than twenty years. This experiment was derived from Molecular Cloning Laboratory Guide (Third Edition), Previous Edition, by Peitang Huang.
Operation method
Polyacrylamide gel electrophoresis purification of oligonucleotide experiment
Materials and Instruments
Sterile filtered water Acetonitrile Ammonium acetate N-butanol Formamide gel spiking buffer without indicator dye Formamide indicator dye mixture Methanol: aqueous solution Oligonucleotide elution buffer TE solution Synthetic oligonucleotide crude product Move makings For more product details, please visit Aladdin Scientific website.
MillexHV Filters Paraffin Sealer Membranes or Fluorescent Thin Layer Chromatography Plates Sep-Pak Traditional Chromatography Columns Syringes UV Lamps Water Bath Heaters
Buffers and solutions
Dilute the cache solution to the appropriate concentration.
Sterile, filtered water (Milli-Q or equivalent) is recommended.
Acetonitrile
Use 10 ml of high performance liquid chromatography (HPLC) grade acetonitrile per Sep-PakC18 column.
Ammonium acetate (10 mol/L)
For each Sep-PakC18 column, use 2 ml of 10 mmol/L ammonium acetate solution.
n-butanol
Formamide gel spiking buffer without indicator dye
Gel Spiking Buffer contains unenriched formamide without indicator dyes [bromophenol blue and/or xylene blue]. Without the addition of the indicator dyes to the Gel Spiking Buffer, the dyes or contaminants in the dyes can move at the same rate as the oligosiderophores and interfere with the detection of the sample by UV absorption (see step 14). If desired, 0.2% Orange Red G can be added to the Gel Spiking Buffer, which migrates to the front of the Spiking Buffer and does not interfere with the detection of the oligos.
Formamide Indicator Dye Mix
This solution is a 50:50 mixture of formamide and an aqueous solution of the indicator dye (0.05% xylene blue and 0.05% bromophenol blue). It can be added to the spiked well adjacent to the oligonucleotide sample as a molecular mass standard.
Methanol: aqueous solution
Mix 6 ml of methanol with 4 ml of filtered sterile water (Milli-Q grade) and use 3 ml of methanol:water solution per Sep-Pak C18 column.
Oligonucleotide Elution Buffer
0.5 mol/L ammonium acetate
10 mmol/L iron acetate
Some researchers have added 0.1% (m/V) SDS to the oligonucleotide elution buffer.This protocol does not recommend the use of SDS for purification of oligonucleotides by Sep-Pak Cl8 column chromatography (see Note to Step 16).
TE Solution (pH 8.0)
Nucleic Acids and Oligonucleotides
Synthetic Oligonucleotide Crude
Synthetic oligonucleotides are usually supplied by the manufacturer as lyophilized products from which the protective groups of the synthesis reaction have been removed (see "Oligonucleotide Synthesis" in the Information section).
Deprotection generally consists of holding the product in a concentrated solution of NH4OH at 55°C for about 5 h. [Some newer DNA synthesis protocols use a different protecting group (e.g., acetylation-protected dC) for one or more nucleosides, which can be removed in less than 10 min].
Prior to purification of the oligonucleotide, it should be determined that the deprotection reaction has been performed. If the oligonucleotide is supplied in NH4OH, 0.5 to 1.0 ml of liquid can be transferred to a 1.5 ml microcentrifuge tube and dried by volatilization at room temperature using a centrifugal dryer (SavantSpeed Vac or equivalent).
When opening a crude oligo for the first time, the cap of the tube should be opened slowly to allow the ammonia to evaporate (it is better to place it in a chemical fume hood), which reduces the chance of the oligo spraying wine around the room.
Specialized equipment
MillexHV filter (Millipore, 0.45nm pore size)
Paraffin sealing membranes or fluorescent thin layer chromatography plates
Chromatography plates are available from Erinkmann (USA) or E. Merck (Europe), e.g. Merck Silica 20 cmX20 cm plates.
Sep-Pak conventional columns, short columns
Sep-Pak conventional columns (available from Waters, Millipore) contain 360 mg/column of hydrophobic ( C18 ) reversed-phase chromatography resin. Purification utilizes the principle that nucleotides bind to the resin in a highly polar solvent (aqueous solution) and elute when the solvent (e.g., methanol-water mixture) decreases in polarity. One column is required for every 10OD160 oligonucleotides in an areal acrylamide gel.
Syringes (5 ml and 10 ml, polypropylene)
One 5 ml and one 10 ml syringe per oligonucleotide sample.
UV lamp (260 nm, portable)
Water bath or preset 55°C heater
Additional Reagents
The reagents required for steps 7 to 9 are listed in Chapter 12, Options 8 and 11.
Methods
Preparation of crude oligonucleotide samples for gel electrophoresis
1. In a sterile microcentrifuge tube, prepare a 10umol/L solution of crude oligonucleotide in sterile filtered water (Milli-Q or equivalent) and shake well.
The solution is often mildly clumpy due to insoluble benzanthracene produced during oligonucleotide synthesis.
2. Centrifuge the solution for 5 min at room temperature at the maximum centrifugal speed of the microcentrifuge, and transfer the supernatant into a new sterile microcentrifuge tube.
3. Extract the solution three times with 400ul of n-butanol (see Appendix 8) to remove the upper organic phase.
If the time is short, the isobutanol extraction can be omitted without causing problems. At this point, centrifuge the solution for 5 min at maximum centrifugal speed at room temperature and transfer the upper sample to a new sterile microcentrifuge tube. Add 10-30ul of the original solution to the 6 spiking slots (lcm) of the denaturing polyacrylamide gel, as described at the end of this chapter.
4. Dry the solution at room temperature using a centrifugal dryer (Savant Speed Vac or equivalent). The centrifuge tube should contain a light yellow precipitate and a milky white powder.
5. Dissolve the precipitate and powder in 200ul of sterile filtered water (Milli-Q or equivalent).
6. Estimate the amount of oligonucleotides in the sample by adding 1ul of solution to 1ml of water, mixing thoroughly and measuring OD260 to calculate the concentration of oligonucleotides.
The amount of oligonucleotide is often expressed in units. 1OD is equivalent to the optical density produced by the amount of oligonucleotide in 1ml of solution in a 1 cm aperture cuvette. The formula for calculating the molar extinction coefficient (e) of an oligonucleotide is given below:
e=A(15.2)+G(12.01)+C(7.05)+T(8.4)
where A, G, C, T are the multiplicity of each nucleotide in the oligonucleotide sequence, and the value in parentheses is the molar extinction coefficient of each deoxynucleotide at pH 8.0. For example, the millimolar extinction coefficient of a 19-polymer oligonucleotide containing 5 dA, 4 dG, 4 dC, and 6 dT residues is
(5X15.2)+(4X12.01)+(4X7.05)+(6X8.4)=202.64 mmol/(L-cm)
Calculate the concentration of undiluted oligonucleotide solution (C) according to the following equation.
C=(OD260)(1000)/s
Gel electrophoresis purification of synthesized oligonucleotides
7. Prepare denaturing polyacrylamide gels (Scheme 8 in Chapter 12) at the appropriate concentration (Table 10-2), with the length of the gel spacer being about 1 cm. 
8. pre-electrophoresis with constant power (50~70W) for about 45 min or gel temperature of 45~50°C. Turn off the power and remove the electrode plug wire.
Pre-electrophoresis allows the ammonium persulfate in the gel wells to be removed, and more importantly, allows the gel to warm up to the ideal temperature for DNA electrophoresis.
9. Without pausing, add approximately 2OD260 of oligonucleotides to one or more of the spiking slots as follows (for highest resolution, the solution volume should be 10ul or <10ul):.
a. Add an equal volume of dye-free formamide spiking buffer to the oligonucleotide solution, mix well with shaking, and heat at 55°C for 5 min to eliminate secondary structure. b. Add the oligonucleotide solution to the sample buffer and mix well with shaking, and heat at 55°C for 5 min to remove secondary structure.
b. Wash out the urea from the spiked wells with 1XTBE.
c. Add heated oligonucleotides to the spiking wells. Add 5ul of dye-containing formamide sample solution to one unused well.
See Chapter 12, Protocol 11 for details on polyacrylamide gel spiking.
10. Electrophoresis at 1500 V until the oligonucleotides have moved to nearly 2/3 of the gel.
The position of the oligonucleotide can be estimated from the position of the dye, as detailed in Table 10-3. It should be noted that synthetic oligonucleotides with a 5'-terminal hydroxyl group move slower on denaturing polyacrylamide gels than phosphorylated oligonucleotides of the same length. Moreover, for unknown reasons, the rate at which an oligonucleotide swims depends on its base composition and sequence. As a result, there is no precise correspondence between the expected and observed positions of oligonucleotides in polyacrylamide gels. 
11. Lay the gel template flat on a test bench matting paper with a plastic protective bottom, with the small piece of plate with the slots up. Cool the gel to below 37°C before proceeding to the next step.
12. Remove the excess tape and slowly and carefully pry off the gel template with a rim strip or gel separator tool, as the gel should be adhering to the longer (unsilanized) glass plate.
Be careful to wear protective restraints as the glass plate may break during this step.
If the gel adheres to two broken glass plates. Put the plates back in place so that the smaller or slotted glass plate has its back to the gel, invert the plates and try again.
13. Place a Sarari membrane over the gel and invert the glass plate so that the gel adheres to the Saraii membrane. Place a paraffin-sealed membrane or fluorescent thin-layer chromatography plate under the gel where the oligonucleotide is expected to be located.
14. Illuminate the gel from above with a portable UV lamp at 260 nm.
The DNA in the gel absorbs the UV light and appears as a dark blue band under the fluorescent back-concentration of the paraffin membrane or fluorescent thin layer chromatography plate. If the DNA bands are difficult to recognize, they can be observed in a dark room with a portable UV lamp.
15. The target oligonucleotide to be recovered should be the slowest migrating band (closest to the tip of the gel). Cut each target DNA band with a sharp, clean scalpel or razor, taking care not to take a UV absorber smaller than the target oligonucleotide.
See "Observation of Oligonucleotides in Polyacrylamide Gels" in the information section at the end of this protocol for additional information on taking the target DNA bands.
16. Transfer the gel slices into 3-4 microcentrifuge tubes, add 1 ml of oligonucleotide elution buffer to each tube, and crush the gel by turning the disposable pipette tip in the tube and pressing it against the wall. The tubes were sealed and incubated at 37°C for 12 h in a shaking incubator.
The SDS-containing elution buffer sometimes produces a milky white precipitate after treatment of Sep-Pak chromatography fractions 2 and 3 after drying. These precipitates are most likely SDS bound to the column, which, when eluted with methanol-water, elutes off the oligonucleotides a little later. As long as the SDS is not co-existing with the oligonucleotides (first component), the decontaminant will not cause problems and may improve recovery. However, even if there is a very small amount of SDS in the final oligonucleotide, it can inhibit subsequent enzymatic reactions (e.g., phosphorylation and primer elongation), which is why this eluate contains SDS.
17. Centrifuge for 5 min at maximum centrifugal speed in a microcentrifuge at room temperature, collect the supernatant, transfer to a 5 ml-disposable syringe, filter through a Millex HV filter, and collect the filtrate in a 15 ml polypropylene tube.
Separation of synthesized oligonucleotides by Sep-Pak C18 columns
18 Prepare the Sep-Pak C18 reversed-phase chromatography column as follows:
a. Attach a 10 ml-secondary syringe barrel to the long end of the Sep-Pak C18 conventional chromatography column.
b. Add 10 ml of acetonitrile to the syringe barrel and slowly push the acetonitrile through the chromatography column with the cartridge.
c. Remove the syringe and pull out the syringe cartridge, which prevents air from being pumped into the chromatography column. Re-attach the syringe barrel to the chromatography column.
d. Add 10 ml of filtered sterile water (Milli-Q grade or equivalent) to the syringe and slowly push with the syringe cartridge to pass the water through the column. Repeat step c.
e. Slowly push 2 ml of 10 mml/L ammonium acetate through the column, remove the syringe, remove the syringe plunger, and reattach the syringe barrel to the column. The column is now ready for use.
19. Add the gel-purified oligonucleotide solution to the syringe barrel (step 17), push the syringe cartridge slowly through the column, and collect the eluate in a sterile 50 ml polyethylene centrifuge tube. Repeat step 18c.
20. Repeat the rinse more than twice by slowly pushing 10 ml of water into the chromatography column with a syringe.
21. Elute the oligonucleotides bound to the Sep-Pak C18 column with lml of methanol-water solution three times, repeating step 18c after each elution. collect the eluates from each elution separately, and measure the OD260 absorbance values of the solutions in each of the three tubes, using the methanol-water solution as a blank. >90% of the oligonucleotides should be present in the first fraction.
22. Volatilize and dry the oligonucleotide-containing solution using a centrifugal dryer.
23. Dissolve the oligonucleotides in a total volume of 200 ul of water or TE (pH 8.0).
24. Aspirate 5ul of solution into a cuvette containing 995ul of water, mix well, measure the OD260 value of the eluted sample, and calculate the total amount of oligonucleotides in the Luo solution as described in step 6 of this protocol (step 23). 
