Array Advice - Labelling

View tips and solutions to common problems for array labelling:

Tips

Labelling the target

  • Always use a closed thermalcycler with heated Lid (105°C).
  • Following denaturation at 99°C for 20 minutes, immediately snap chill samples on wet ice or cold blocks. Do not leave in thermalcycler to ramp down to 4°C.
  • Take care when pipetting the viscous Klenow enzyme.
  • Avoid evaporation from PCR tubes by ensuring a good seal on caps.
  • When adding reagents to the sample, ensure efficient mixing either by rapid pipetting or gentle vortexing. Failure to mix the reagents and samples properly could lead to inefficient sample labelling and dye incorporation or uneven hybridisation of the labelled target to the arrays.

Purifying the labelled target

  • Check clean up columns are spun at the correct centrifuge speed - 2000g for 1 minute.
  • Add labelled DNA to the centre of the gel matrix in your purification plates or columns to ensure efficient purification.

Solutions to common problems

Problem - Low dye incorporation

Causes:

  1. Labelling master mix incorrectly prepared- incorrect volume of reagents/no reagent added, insufficient mixing.
  2. Incorrect heat fragmentation/labelling temperature/time.
  3. Excessive exposure to light or air.
  4. Loss of solution from evaporation.

Solutions:

  1. Ensure that each reagent is added.
  2. Check that the correct volumes are added (pipettes are calibrated).
  3. Gently vortex reagent tubes except Klenow.
  4. Gently flick master mix tubes and quick spin.
  5. Ensure thermocycler block is at the correct temperature.
  6. Ensure the heat fragmentation time is exactly 20 minutes.
  7. Use a thermocycler with heated lid to prevent excessive exposure to light/air.
  8. Make sure the sample tubes/plate wells are closed/sealed properly.

 

CytoSure™ products are for research use only; not for use in diagnostic procedures