LPA Troubleshooting for Uneven Staining

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, uneven staining can occur due to the following reasons:

• Strips not completely immersed in reagents during incubation steps​.
• Tray not shaken properly. 
• Denatured amplicons were not properly mixed with the hybridization buffer before adding the strip to the well.
• The reusable tray had not been properly cleaned so residual amplicons remained.

Solution: Repeat reverse hybridization step​.

Figure: Uneven Staining of the Strip

Points to Note

• The strips must be checked to ensure that they are fully immersed after the addition of each reagent. If necessary, a pipette tip can be placed on top of a strip to keep it submerged. Care must be taken so that buffer from a well with a problematic strip does not spill into any of the other wells.
• Before adding the strip to the well, the purple solution containing the denatured amplicons should be completely diffused into the green hybridization buffer.
• The instruments should be checked to ensure that the Twincubator is shaking at 300 rpm and the GT-Blot 48 is oscillating properly.
• The trays must be thoroughly washed and rinsed after each use.

Do’s and Don’ts

• Gently shake the tray until the solution has homogenous colour.
• Do not spill solution into the neighboring wells.
• Tray must be dipped into the water to at least 1/3rd of its height.
• Use washed and clean trays.

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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