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There could be overall weak or no signals on the strip including or except for the conjugate control zone.

 

Weak or no signal​ including conjugate control (CC) zone could be due to:

 

  • Too low room temperature
  • Reagents not equilibrated to room temperature (20-22°C)
  • Hybridization solutions not pre-warmed
  • Too high or too low incubation temperature
  • No or too little amount of CON-C and/or SUB-C used

 

Solution: Repeat reverse hybridization.

 

Figure: No Signal including CC Zone

 

 

Points to Note

 

  • A higher incubation temperature may prevent the binding of the amplicons to the probes.
  • A higher temperature during the stringent wash step may result in weak intensities of the banding patterns.
  • The temperature of the hybridizer should be monitored if such problems are seen.
  • If the problem persists and the thermostat on the hybridizer gives an appropriate reading of 45ºC, and a digital thermometer reads a different temperature that is above or below 45ºC; the problem is likely with the machine’s internal temperature probe and should be reported to the manufacturer and corrected.

 

Do’s and Don'ts to Avoid Weak or No Signals

 

  • Perform LPA at the appropriate room temperature.
  • Bring reagents to room temperature before use (20-22°C).
  • The green and red hybridization solutions must be pre-warmed to 45ºC.
  • Follow instructions to add the exact volume of reagents.
  • The central heating/cooling thermostat must be appropriately adjusted.
  • Do not use pipettes that are not calibrated.
  • Do not miss any step in the hybridization process.

 

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