Review selected protocols that are commonly used to spectrophotometrically quantify the concentration of an oligonucleotide or primer solution; how to concentrate or precipitate an oligo; how to PAGE purify oligos; or how to build adaptors with your oligos.
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 = 0.001 L
24 nmole/0.001 L = 24000 nmole/L or 24000 nM
24000 nmole/L X 1 µmole/1000 nmole = 24 µmole/L or 24 µM
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 µmole to get the µM conc.
Example 2: making 100 µM primer stock:
If COA specifies we have 24 nmole of oligo:
24 nmole X 1 µmole/1000nmole = 0.024 µmole
0.024 µmole/100 µmole/liter = 0.00024 L
0.00024 L X 1000 ml/L = 0.24 ml or 240 µl
Explanation: We convert from nmole to µmole then divide by the desired concentration in µmole/L. The µmoles 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 µM solution.
Example 3: Calculate from OD. If primer OD is 0.14:
If the µg/OD reported on the COA is 36.6:
0.14 OD/ml X 1000 µl/10 µl = 14 OD/ml stock
14 OD/ml X 36.6 µg/OD = 512.4 µg/ml
Explanation: The OD/ml is multiplied by the dilution factor to get the stock OD/ml. The OD is converted to µg/ml by multiplying the OD/ml of the stock by the µg/OD conversion factor listed on the COA. The µg cancel out giving µg/ml.
Example 4: Calculate from MW. The COA specifies a molecular weight for the oligo of 7440.0:
7440.9 g/mole = 7440.9 µg/µmole
7440.9 µg/µmole X 68.6 µmole/L = 510445.74 µg/L
510445.74 µg/L X 1 L/ 1000 ml = 510.4 µg/ml
Explanation: g/mole is the same as µg/µmole. The molecular weight expressed in µg/µmole is multiplied by the µM concentration determined in example 3. The µmole cancel out leaving µg/L. Liters are converted to ml to give the µg/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.
(Protocol excerpt from Invitrogen GeneTrapper Manual. Note: The resolution is poor on precast mini polyacrylamide gels of smaller (i.e. >25 nt) oligos.)
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 oligonucleotides 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|
For Research Use Only. Not for use in diagnostic procedures.