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During hybridization, single-stranded amplicons bind to probes and highly specific binding is ensured to produce even staining in the presence of stringent buffer and temperature conditions. However, high/ dark backgrounds in strips may be due to the following reasons:

 

  • CON-C and/or SUB-C used too concentrated​
  • Washing steps not performed with necessary care
  • Wash solutions were too cold​

 

Solution: Repeat reverse hybridization step.​

 

Figure: Dark background in the LPA Strip

 

 

Points to Note

 

  • The rinse step should be done carefully to remove excess conjugate buffer followed by a wash with deionized water to remove salts in the rinse buffer.
  • Post-substrate development, wash with deionized water is essential to stop colorimetric reaction and to remove excess substrate buffer.
  • Reagents should always be warmed before use.

 

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