LPA Troubleshooting for High Background Colour

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.

Resources

• GenoType MTBDR plus ver 2.0 Kit, Instructions for Use.
• GenoType MTBDRsl VER 2.0 Kit, Instructions for Use.
• GLI Training Package on LPA.

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