Protocols

Reconstitution and Storage

  • Spin tube briefly in microcentrifuge to collect DNA in bottom of tube.
  • Add desired amount of buffer to tube (TE, 10mM Tris pH 8.0 or DNase-free water recommended). We recommend resuspending at a high concentration and diluting to a working stock when necessary.
  • Allow to rehydrate for a few minutes with intermittent vortexing.
  • Once resuspended, store at -20°C.
  • Avoid multiple freeze/thaw cycles by aliquoting before freezing.
  • Only resuspend one tube at a time (if two or more tubes are supplied for a single oligo).
  • Store fluorescent primers and probes in the dark

To Quantify by Spectrophotometer

GeneWorks oligos are quantified before lyophilisation. The label states the amount per tube in µg and nmoles and the molecular weight and Tm of the oligo. The data sheet includes the Extinction Coefficient (E260) for the oligo.

To further quantify a solution of oligo:

  1. Dilute an aliquot of resuspended oligo in water to a final volume of 1000µl
  2. Calculate dilution factor (eg: if 50µl diluted, the dilution factor would be 1000/50=20)
  3. Vortex
  4. Read absorbance at 260 nm

Approx. Stock Conc. (µg/ml) = A260 x dilution factor x 30

Stock Conc. (M) = (A260 x dilution factor) / E260

NB: Assumes use of a 1 cm path length cuvette.

Conversions

To convert µg to µmoles (Note: 1g=103mg=106µg=109ng=1012pg):

µmoles of oligo = µg oligo / MW of oligo

To convert to molarity, use the following equation, where µl is the amount used to resuspend the oligo:

M = µmoles / µl

To determine how much of your stock to add to a reaction, first determine what the final oligo concentration should be, then use the following equation:

Volume (stock) x Conc. (stock) = Volume (rxn) x Conc. (rxn)

Ethanol Precipitation of Oligos

Generally, ethanol precipitation of GeneWorks oligos is not required. However, if it is necessary for sensitive applications, or in case of accidental over-dilution, it can be done. It may be safer to add a carrier to the oligo such as glycogen or tRNA if the oligo is very small, or in very small quantities. One recommended procedure is:

  1. Accurately determine the volume of the oligo
  2. Add 1/10th volume of 3 M sodium acetate (pH 5.2)
  3. Add 2.5 volumes of cold absolute ethanol
  4. Leave at -20°C for >30 minutes
  5. Centrifuge at top speed for 20 min at 4oC
  6. Carefully remove the ethanol, without disturbing the pellet
  7. Wash pellet with 0.5 ml cold 70% ethanol
  8. Centrifuge briefly
  9. Carefully remove supernatant

Allow to dry before freezing or resuspending.

It is recommended that the supernatant from step 6 is retained until the final oligo concentration after precipitation is determined. Other precipitation methods may also be used, such as using ammonium acetate instead of sodium acetate.

Making dsDNA by OIigo Annealing

Dissolve both oligos (complementary strands of duplex) to the same molar concentration (typically 10-100 µM) in buffer of choice eg 10mM Tris or TE pH 8.0.

Mix together equal volumes of the oligo solutions. Heat to 95ºC for 5 minutes then allow to cool gradually to room temperature over approximately 30 minutes.

The efficiency of annealing can be determined if desired by running the individual and annealed oligos in neighbouring tracks on a non-denaturing acrylamide gel (acrylamide concentration between 15 and 20%). DNA can be visualised by staining with ethidium bromide or SBYR Gold.

Treatment of Thiol labelled oligos with DTT solution

As oxidative disulfide formation occurs with thiol-modified oligos, they should be reduced before use.
The oligo can be treated with DTT prior to use, or stored in DTT solution for convenience. In both cases, DTT must be removed immediately before the oligo is used in a conjugation reaction.

  1. Make a 100 µM solution of the oligo in TE (10 mM Tris-HCl, 1 mM EDTA pH 8.0) containing 10 mM DTT.
  2. Pass the oligo solution through a large bed volume gel filtration column (eg Sephadex) to remove DTT. The column can be pre-equilibrated with your buffer of choice (eg conjugation reaction buffer) if desired. The presence and concentration of oligo in the eluate should be confirmed by UV absorbance spectroscopy at 260 nm.

Traces of DTT remaining with the oligo can interfere with subsequent conjugation. The use of small bed volume or spin gel filtration columns is therefore not recommended.

It is possible to store reduced oligos frozen immediately after removal of DTT. However since re-oxidation will gradually occur re-treatment may be necessary before use.

 

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