Posted on May 16th, 2016 by UNM CC
Stocks should be handled with care to ensure that they are not contaminated with PCR product or plasmid DNA that might be prevalent in the laboratory. For this reason they are handled only in the pre-PCR area with gloves, aerosol resistant tips, and DNAse/contamination-free solutions.
Oligo concentrations are typically expressed in terms of moles, not weight (µg). The extinction coefficient, for converting absorbance (A260) to concentration (µM), varies according to the base composition of the oligo. In this protocol, the MPD is used to record the oligo sequence, to calculate its extinction coefficient and concentration, and to note the location of the freezer stock solution.
- DNAse free H2O or Tris 10mM, pH=8
- 1.5 mL Eppendorf tubes
- Aerosol resistant pipet tips
- Oligonucleotide stock (from manufacturer)
- Turn on spectrophotometer.
- Take bag containing oligo to pre-PCR room. Dump tube on bench, without touching tube or inner surface of bag.
- Don gloves. Place tube in pre-PCR rack.
- Add 10 µL of H2O or 10mM tris per nMole of stock primer (quantity according to the oligo maker). Vortex.
- Dilute Oligo 1:50 v:v by adding 10 uL to 490 uL H2O. Also aliquot 500 uL H2O as a blank.
- Using a glass cuvette (not plastic) blank the spec. using the H2O. Measure A260 of the oligo.
- Find or enter the oligo name in the MPD on the Oligo Details layout. Enter the A260 value and dilution factor (50).
Record the concentration of the oligo in your notebook.
- In the pre-PCR room don fresh gloves and write the concentration of the oligo on the stock tube.
- In a 1.5 mL tube, dilute the primer stock down to the working concentration of 10 uM with H2O or 10 mM Tris, pH8.
- Place the remaining oligo stock at -80ºC, and record it's location in the MPD.