Nick Translation DNA labeling

Technical Support Request Form

Low yield

• Poor quality template DNA >> purify the template DNA
  
• Overdigestion with DNASE I >> use less DNase I
  
  

Probe fragments too long (> 800 bp = punctuate background)

• Incorrect temperature >> Move to calibrated waterbath or thermocycler
  
• Not enough DNase I >> increase the amount of DNase I or increase the reaction time
  
• Inaccurate quantitation >> Not enough DNase I, add more and incubate additional 30 minutes
  
  

Probe fragments too small (<100 bp = low signal, diffuse background)

• Inaccurate quantitation due to poor quality template >> Purify template DNA
  
• Degraded template >> Purify template DNA or increase the amount of template DNA in reaction
  
  

High background

• Inadequate hybridization conditions >> Use tightly sealed chamber with appropriate humidity control.
  
  

Low Signal

• Inadequate denaturation of slide or probe >> Increase denaturation temperature to 74°C
  
• Inappropriate filter set on microscope >> Multiple bandpass filter sets provide less light than single bandpass. Use correct filter or fluorophore.