西亚试剂：：Oligo - Storage and Handling

and Handling

Use of oligonucleotides in various research applications requires certain basic storage and handling techniques in order to ensure trouble-free experiments. Proper storage of your oligonucleotide will maximize its shelf-life, allowing you to get the most use from your oligo. Following these simple dilution and weight/volume unit conversion guidelines will enable you to work easily with your Sigma-Genosys oligos.

What should I use to resuspend my oligo?

Oligonucleotides may be resuspended in either sterile water or, preferably, TE (10mM Tris, pH 7.5-8.0, 1 mM EDTA). The use of DEPC-treated water is not recommended. If you are making a concentrated stock solution we recommend dissolving your oligo in TE. Working solutions can be diluted from the stock solution using sterile, nuclease-free water.

How should I store my oligo?

All Sigma-Genosys oligos are provided desalted, dry, and ready for use upon resuspension. Once resuspended, oligonucleotide stock solutions are best kept frozen at -20篊 for several weeks and may remain stable for several months. The most important factor in storing working solutions is using nuclease-free, sterile water. For long-term storage of oligonucleotide stocks, we recommend that you dry down the samples and store at -20篊. Oligonucleotides modified with fluorescent dyes should be kept away from light as much as possible. Alkaline phosphatase modified oligonucleotides should be stored at 4篊, never frozen.

How did Sigma-Genosys quantify my oligo?

The microgram quantity of oligonucleotide we supply is directly related to its UV optical density (O.D.) at a wavelength of 260 nm. Sigma-Genosys takes great care to ensure that the O.D. reading determined for each oligonucleotide is as accurate as possible. After desalting and further purification of your oligo we resuspend it in 200 ul of water. We remove a 6 ul aliquot (to ensure we are taking a volume within the accurate and reproducible range of micropipettes) from the suspension and dilute it with 994 ul of sterile water required to determine the O.D. using a spectrophotometer. We ensure that the O.D. reading is within the linear range of accuracy of our spectrophotometers. Any sample not within 0.1 and 1.5 O.D. units is remeasured using a more appropriate dilution for that sample.

Because we take only 6 ul from the original 200 ul resuspension, the O.D. of the 6 ul must be multiplied by a factor of 33 (which is 200/6) to determine the O.D. of the whole resuspension. In general, it can be assumed that 1 O.D. unit is equivalent to 30 ug, which is a reasonable average for most oligonucleotides. Therefore, the total number of micrograms of oligo in the 200 ul resuspension can be calculated as follows:

Total ug of oligo in original 200 ul

O.D. reading for X 33 X 30 ug
6 ul aliquot

Sigma-Genosys accurately determines the extinction coefficient for each oligo (see ) The quantity of oligonucleotide sent to you (see) is expressed in nanomoles on your tube label, along with the ug quantity of the oligo and its molecular weight (MW). We calculate the number of nanomoles using the amount of oligo (in ug) and the molecular weight as follows:

Total nmole oligo	=	ug of oligo	X	1 umol	X	1000 nmole
				MW		1 micromole
	=	ug of oligo	X	1000
				MW

(Note: 1 mole=10e3 mmoles=10e6 micromoles=10e9 nanomoles=10e12 picomoles)

Sample Research Application

"My tube contains 35.22 nmoles of oligo, but I need a final oligonucleotide concentration of 0.5 uM in my reaction. My reaction volume is 100 ul. How much oligo do I add to my reaction?"

First, make a concentrated oligonucleotide stock solution (10X or 100X, depending on the concentration you need). Then add an appropriate volume to the reaction, thereby achieving the desired working concentration. Serial dilutions of the stock(s) may be required to ensure pipetting accuracy. See outlined example below:

1. Decide on a stock concentration and standardize your units.

	Desired final concentration = 0.5 uM
	100X stock concentration = 100 X 0.5 uM = 50 micromole/liter
	Total nanomoles of oligo = 35.22 nmole (see tube label for your actual oligo)
	Total micromoles of oligo = 35.22/1000 = 0.03522 micromole
	Note: The quantity of oligo must be expressed in micromoles to calculate the volume for a micromolar solution.

2. Make a 100X stock solution (100 X 0.5 uM = 50 uM).

What volume (V) is required to resuspend 0.03522 micromole of oligo to give a stock concentration of 50 uM? Use the following steps as a guide:

	50 micromole	=	0.03522 micromole	Solve for V
	1000 ml		V ml

	V	=	0.03522 x 1000 m
			50

V = 0.704 ml = 704 ul

3. Use serial dilutions to make a 10X stock (10X 0.5 uM = 5uM).

For pipetting accuracy, avoid using small volumes (less than 2 ul) of stock solutions in your reactions. It may be necessary, therefore, to serially dilute your stock oligonucleotide solution. Make a 10X stock by diluting your 100X stock as follows:

1:10 dilution of 100X stock

10 ul of 50 uM (100X) stock + 90 ul sterile water (or TE) = 100 ul of 5 uM (10X) stock

4. Use a 10X stock to give the final concentration in the reaction.

My reaction volume is 100 ul. How much of the 10X (5 uM) stock solution of oligo is required to yield a 0.5 uM concentration of oligo in the total reaction?

Vol. of (10X) stock X stock conc. = Final reaction vol. X final oligo conc.

(S ul) X (5 uM) = (100 ul) X (0.5 uM) Solve for S

S	=	100 X 0.5 ul
		5

S = 10 ul