Protocols for Oligonucleotides
1. Protocol for the Quantitation of Oligonucleotides, Spectrophotometrically:
Add an aliquot of the resuspended oligonucleotide to a final volume of 1,000 µl with water (water = 1,000 µl – volume of oligonucleotide added). Vortex or pipette up and down for 15 seconds. Read the absorbance of this dilution at 260 nm (A260). Use the formula below to calculate the concentration (use the calculated Weight per OD from the Certificate of Analysis in your calculation) of oligonucleotide in your stock solution.
Concentration in µg/ml = A260 × Weight per OD × dilution factor*
(* the dilution factor is determined by 1000/amount of resuspended oligo added for the dilution, ie., if you added 50 µl of resuspended oligo to 950 µl of water to read the absorbance, you dilution factor would be 1,000/50 = 20)
Example 1: the COA specifies we have 24 nmole of oligo. If we resuspended oligo in 1 ml: 1 ml = .001 L
24 nmole/0.001 L = 24000 nmole/L or 24000 nM
24000 nmole/L X 1 umole/1000 nmole = 24 umole/L or 24 uM
Explanation: We convert the volume in which the oligo was resuspended into liters. Then the total nmole amount of oligo is divided by the volume to get nM conc. The nmoles are converted to umole to get the uM conc.
Example 2: making 100 uM primer stock:
If COA specifies we have 24 nmole of oligo:
24 nmole X 1umole/1000nmole = 0.024 umole
0.024 umole/100 umole/liter = 0.00024 L
0.00024 L X 1000 ml/L = 0.24 ml or 240 ul
Explanation: We convert from nmole to umole then divide by the desired concentration in umole/L. The umoles cancel out giving the needed volume in liters. We then convert liters to ml. So in this example the oligo should be suspended in 0.24 ml to get a 100 uM solution.
Example 3: Calculate from OD. If primer OD is 0.14:
If the ug/OD reported on the COA is 36.6:
0.14 OD/ml X 1000ul/10ul = 14 OD/ml stock
14 OD/ml X 36.6 ug/OD = 512.4 ug/ml
Explanation: The OD/ml is multiplied by the dilution factor to get the stock OD/ml. The OD is converted to ug/ml by multiplying the OD/ml of the stock by the ug/OD conversion factor listed on the COA. The ug cancel out giving ug/ml.
Example 4: Calculate from MW. the COA specifies a molecular weight for the oligo of 7440.0:
7440.9 g/mole = 7440.9 ug/umole
7440.9 ug/umole X 68.6 umole/L = 510445.74 ug/L
510445.74 ug/L X 1 L/ 1000 ml = 510.4 ug/ml
Explanation: g/mole is the same as ug/umole. The molecular weight expressed in ug/umole is multiplied by the uM concentration determined in example 3. The umole cancel out leaving ug/L. Liters are converted to ml to give the ug/ml concentration.
For most purposes, ethanol precipitation is not required prior to use. However, if ethanol precipitation is desired the following protocol may be used for oligonucleotides > 20 bases in length and a quantity > 0.1 OD. For very small amounts of material a carrier such as tRNA should be used.
- Dry the oligonucleotide in a microcentrifuge tube.
- Redissolve the pellet in 0.2 ml of 0.3 M Sodium Acetate (pH 7.0).
- Add 0.6 ml cold absolute ethanol (-20°C) and place at 4°C for 30 minutes.
- Centrifuge at 10,000 × g for 30 minutes.
- Carefully remove the supernatant without disturbing the pellet.
- Add 1 ml of cold 70% (v/v) ethanol.
- Centrifuge at 10,000 × g for 10 minutes.
- Carefully remove the supernatant (without disturbing the pellet) and evaporate to dryness.
- Store the oligonucleotide at -20°C.
The resolution is poor on precast mini ployacrylamide gels of smaller (i.e. < 25 nt) oligos.
- Use a 1.5 mm gel with well forming combs (not sharkstooth combs).
- Prepare a 12% acrylamide gel (19:1) with 8 M urea and 1X TBE.
- Dissolve a 5-10 OD of oligo (approx. 1 OD = 33 µg) in 25 µl TE. Add equal volume of formamide to the oligo and heat to 95°C for 1 min. Chill on ice. To avoid masking the location of the oligo (when detecting by UV shadowing) do not add dye to the formamide. We suggest an outside lane not containing oligo have the dye added for tracking purposes.
- Flush wells before loading oligos. Always leave a space between lanes containing different oligos. Electrophorese 20-30 cm. For the Model SA or Model S2 electrophorese at ~60W or 600-700 V. For the Model V16 or V15.17 electrophorese at 300 V.
- Place gel on a piece of plastic wrap and then on an X-ray intensifying screen (intensifying side up towards gel). Examine gel with short wave UV light from above (w/o EtBr staining). The oligo band(s) will appear as a dark shadow.
- Excise the areas of the gel containing the full length oligo. Crush the gel slice and elute the oligo in 1 ml TE overnight at 37°C with shaking.
- Transfer the eluted solution to a fresh tube with a 1 ml pipette. Wash the gel slice with a 200 µl of TE and combine with the eluted solution. Measure the total volume of the eluted oligo.
- After washing a PD 10 column with 12 ml of autoclaved water, add the eluted oligonucleotide to the column and discard the flow through.
- Wash the column with autoclaved, distilled water, using a volume equal to 2.5 minus the total volume of the eluted oligonucleotide which was loaded onto the column. Discard the wash flow through.
- Elute the oligonucleotide by adding 1 ml of autoclaved, distilled water to the column and collect this fraction in a sterile tube. Add another 1 ml of autoclaved distilled water and collect it in a second tube.
- Dry the oligo under vacuum.
- Dissolve the oligo in 100 µl of TE.
- Add 100 µl of Phenol: Chloroform: Isoamyl and vortex thoroughly. Spin at RT for 5 min at 14,000 x g. Remove 90 µl of upper aqueous layer and transfer it to a fresh tube.
- Add 45 µl of 7.5 M NH4OAC and 350 µl absolute EtOH. Vortex to mix, store on ice for 10 min and spin for 10 min at 14,000 x g at 4°C.
- Remove supernatant, wash pellet with 70% EtOH.
- Dry oligo and dissolve in 60 µl TE. Verify by OD that oligo concentration is greater than 0.5 µg/µl.
5. Protocol for Adapter Production/ DNA Duplex:
It is recommended that oligos be PAGE purified for full length prior to use in building adapters. Alternatively PAGE purified, cartridge purified or HPLC purified primers that are commercially available are acceptable.
Verify the DNA concentration by preparing duplicate dilutions and determining the A260. Amount of oligo (nmol) = A260 x nmole per OD x dilution factor
To prepare the adapter, add the following components together: (You will need to calculate the volumes based on the concentrations of the oligonucelotides and total volume of the annealing reaction. You may scale the reaction to suit your needs).
|Oligonucleotide 1||100 nmol|
|Oligonucleotide 2||100 nmol|
|10X Annealing Buffer *||1 X DEPC-treated water to appropriate volume|
Bring the oligo solution to 65° by placing the tube in a 65°C or higher water bath. Maintain the oligo solution at exactly 65°C for 10 minutes. (It is critical to maintain the oligo at exactly 65° for the duration of this time). Remove the solution from the water bath and allow it to cool slowly at room temperature (1-2 hours).
Store the adapter at -20°C.
*10X Annealing Buffer:
Mix the following components for a 50 ml stock solution:
|1 M Tris-HCl (pH 7.5)||5 ml||100 mM|
|5 M NaCl||10 ml||1
|0.5 M EDTA||1 ml||10 m|
|DEPC-treated water||34 ml|