Technical Support
by Application Area

The most common causes of no-band results are:

• Primer failure — check Tm, GC content (40–60%), and that primers don't form dimers
• Degraded template — run template on gel to confirm integrity; A260/A280 should be 1.8–2.0
• Inhibitors in template — carry-over of ethanol, phenol, or EDTA can block polymerase
• Incorrect Mg²⁺ concentration — standard is 1.5 mM; titrate 1.0–3.0 mM
• Annealing temperature too high — try reducing by 5°C increments

Non-specific amplification is usually caused by low annealing stringency or mispriming.

• Raise annealing temperature in 2°C steps until non-specific bands disappear
• Use a hot-start polymerase to prevent extension at room temperature
• Reduce template concentration — over-loading drives non-specific products
• Add 5% DMSO or betaine for GC-rich targets

For most qPCR assays, 1–10 ng of cDNA or genomic DNA per 20 µL reaction is optimal. Over-loading (> 100 ng) causes inhibition and artificially high Cq values. Under-loading (< 0.01 ng) increases inter-replicate variability.

Always run a standard curve across 4–5 log dilutions to verify reaction efficiency (90–110% acceptable). Efficiency is calculated as: E = (10^(–1/slope)) – 1.

Low signal typically points to one of these causes:

• Reagent expiry or freeze-thaw damage — check lot dates; avoid repeated freeze-thaw of antibodies
• Insufficient incubation time or temperature — ensure plate is at correct temperature and incubation not cut short
• Substrate not activated — TMB substrate should be at room temperature before use
• Sample concentration too low — ensure sample is within the standard curve range; concentrate if necessary

Mycoplasma is the most prevalent cell culture contaminant and is invisible to the eye or standard bright-field microscopy. Detection methods:

• PCR-based detection — most sensitive; ABMIUM Mycoplasma Detection Kit (Cat. AB-MYC-01) detects > 90 species
• DAPI/Hoechst staining — mycoplasma appear as small fluorescent dots around nuclei

For elimination, BM-Cyclin treatment over two 7-day cycles is effective for most strains. Confirm clearance 2 weeks after treatment.