FAQ
common troubleshooting problems and solutions for PCR and qPCR
No PCR product or weak amplification
Check DNA quality and quantity (use 50-100 ng of template DNA).
Optimize primer design (ensure no secondary structures or dimers).
Adjust annealing temperature (use a gradient PCR to find the optimal temperature).
Increase the number of cycles (25-35 cycles is typical).
Verify polymerase activity and Mg²⁺ concentration.
Non-specific bands or smearing
Increase annealing temperature to improve primer specificity.
Use hot-start Taq polymerase to prevent non-specific amplification.
Optimize Mg²⁺ concentration (too much can cause non-specific binding).
Reduce the number of cycles to minimize by-products.
Check primer design for potential off-target binding.
Primer-dimer formation
Redesign primers to avoid complementary sequences at the 3′ ends.
Increase annealing temperature.
Reduce primer concentration (typically 0.1-0.5 µM is sufficient).
Use a hot-start polymerase to prevent early primer extension.
Low yield of PCR product
Increase the amount of template DNA.
Optimize extension time and temperature for the target length.
Check polymerase activity and buffer composition.
Ensure primers are not degraded (store at -20°C and avoid repeated freeze-thaw cycles).
Common Troubleshooting in Real-Time PCR : No amplification or late Ct values
Check template quality and quantity (degraded or insufficient template can cause issues).
Optimize primer concentrations (typically 0.1-0.5 µM).
Verify primer specificity and design (avoid secondary structures or dimers).
Ensure proper reaction conditions (e.g., annealing temperature, Mg²⁺ concentration).
Non-specific amplification or multiple peaks in melt curve (SYBR Green)
Redesign primers to improve specificity.
Increase annealing temperature.
Use a hot-start polymerase to prevent non-specific binding.
Optimize Mg²⁺ concentration.
High background fluorescence (TaqMan Probes)
Ensure probes are not degraded (store properly and avoid repeated freeze-thaw cycles).
Check for probe-primer interactions (redesign if necessary).
Optimize probe concentration (typically 0.1-0.2 µM).
Best Practices for Real-Time PCR
Use High-Quality Templates: Ensure DNA/RNA is pure and free of contaminants.
Optimize Primer and Probe Design: Use software to avoid secondary structures and ensure specificity.
Include Controls:
No-template control (NTC): Detects contamination.
Positive control: Ensures reaction is working.
Housekeeping gene: Normalizes gene expression data.
Calibrate Instruments Regularly: Ensure accurate fluorescence detection.
Analyze Data Carefully: Use appropriate software and statistical methods.
FAQ
Restriction Enzyme Digestion Troubleshooting
1. Incomplete or no digestion
Check enzyme activity: Ensure the enzyme is not expired or degraded (store at -20°C and avoid repeated freeze-thaw cycles).
Verify reaction conditions: Use the correct buffer, temperature, and incubation time specified by the manufacturer.
Check DNA quality: Ensure the DNA is pure and free of contaminants (e.g., phenol, ethanol, or salts).
Inhibit star activity: Avoid excessive enzyme amounts (typically 1 unit per µg of DNA) and ensure proper buffer conditions.
2. Unexpected band sizes on the gel
Verify enzyme specificity: Ensure the enzyme recognizes the correct sequence and cuts at the expected sites.
Check for methylation: Some enzymes are sensitive to DNA methylation (use methylation-insensitive enzymes if needed).
Confirm DNA concentration: Overloading the gel can cause smearing or distorted bands.
Look for partial digestion: If bands are faint or incomplete, increase incubation time or enzyme amount.
3. Star activity (non-specific cutting)
Reduce enzyme amount: Use the recommended amount of enzyme (1 unit per µg of DNA).
Optimize buffer conditions: Use the correct buffer and avoid high glycerol concentrations (>5%).
Shorten incubation time: Prolonged incubation can increase star activity.
Lower reaction volume: Increase DNA concentration to reduce enzyme-to-DNA ratio.
4. Poor ligation efficiency after digestion
Verify complete digestion: Run a gel to confirm the DNA is fully cut.
Dephosphorylate the vector: Prevent self-ligation by dephosphorylating the vector ends.
Purify digested DNA: Remove enzymes and salts using a PCR purification kit or gel extraction.
Check insert-to-vector ratio: Use a 3:1 molar ratio of insert to vector for sticky ends and 1:1 for blunt ends.
5. Enzyme does not cut the DNA
Check recognition sequence: Ensure the DNA contains the correct restriction site.
Test enzyme activity: Use a control DNA with a known restriction site to confirm enzyme functionality.
Inhibit enzyme inhibitors: Ensure the DNA is free of contaminants like EDTA, SDS, or ethanol.
Verify reaction setup: Double-check buffer, temperature, and incubation time.
6. Multiple bands or smearing on the gel
Check DNA quality: Degraded or contaminated DNA can cause smearing.
Optimize enzyme amount: Too much enzyme can lead to non-specific cutting.
Reduce incubation time: Overdigestion can cause additional cuts or degradation.
Use fresh reagents: Old or improperly stored enzymes and buffers may cause issues.
Best Practices for Restriction Enzyme Digestion
Use High-Quality DNA: Ensure DNA is pure and free of contaminants.
Follow Manufacturer’s Guidelines: Use the recommended buffer, temperature, and enzyme amount.
Include Controls:
Uncut DNA control: Verify DNA integrity.
Control DNA with known sites: Confirm enzyme activity.
Avoid Star Activity: Use the correct buffer, minimize glycerol, and avoid excessive enzyme.
Verify Digestion: Always run a gel to confirm complete digestion before proceeding to ligation or other steps.