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LPA Troubleshooting for High Background Colour
Learning ObjectivesLPA Troubleshooting for High Background Colour
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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.
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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