Those who have done PCR know that mastering this technique and becoming a true PCR expert is no easy task.  When it comes to qPCR, it seems like every single step can ruin the entire experiment, making it a constant headache for seasoned researchers.  Sometimes, issues like false positives, nonspecific bands, and other problems just won’t go away—what should you do then?  Practice makes perfect, so we’ve compiled the hard-earned wisdom from experienced scientists to share with you.

Q1: False positives in PCR products (i.e., the negative control shows target amplification)

Answer：

1. Poor primer design: If the primers have homology with non-target sequences, PCR may amplify unintended products.    Additionally, if the target sequence or primers are too short, false positives may occur.    In such cases, redesign the primers.

2. Preventing cross-contamination from genomic DNA or large DNA fragments, Handle samples gently to avoid aerosol contamination (e.g., pipetting target sequences into the reaction or splashing them outside the tube);  All reagents and equipment (except enzymes and heat-sensitive materials) should be sterilized at high temperatures, Use disposable centrifuge tubes and pipette tips; If necessary, expose reaction tubes and reagents to UV irradiation before setup to degrade any contaminating nucleic acids.

3. Preventing contamination from small airborne nucleic acid fragments (with partial homology to the target sequence), Nested PCR can be used to reduce or eliminate such contamination.

Q2: False negatives or no amplification in PCR products (i.e., bands appear in the positive control but not in the sample)

Answer:

1.  If the strips are left for too long, the nucleic acid may degrade.  It is best to perform electrophoresis detection within 48 hours.

2.  Low purity of DNA template (e.g., containing impurities, proteins, or Taq enzyme inhibitors): The DNA can be repurified or re-extracted using a high-quality kit.  If the DNA concentration is too low, increase the template amount.  For DNA with secondary structures, use a better polymerase.  When extracting DNA, avoid inhaling phenolic reagents.

3.  For poorly designed primers, redesign and resynthesize them.  Primers should be stored in small aliquots at high concentrations to prevent degradation from repeated freeze-thaw cycles.  Measure the OD value of the primers and perform electrophoresis to ensure equal concentrations of both primers.

4.  If the enzyme is inactive, replace it with a new one or use a combination of old and new enzymes to determine whether the false-negative result is due to loss or insufficient enzyme activity.

5.  PCR reaction conditions: Increase the denaturation/annealing temperature;  appropriately increase the number of cycles.

6.  If the Mg²⁺ concentration is too low, it may reduce PCR amplification yield or even cause amplification failure.  Conversely, excessive Mg²⁺ concentration can reduce PCR specificity.  Therefore, appropriately adjusting Mg²⁺ concentration is recommended.

7.  If mutations or deletions occur in the target sequence, they may also affect the specific binding between primers and the template, leading to false-negative results.

Q3: Non-specific band amplification or smearing of bands

Answer：

1. If primer specificity is poor or primer dimers form, redesign the primers or use nested PCR.

2. If template or primer concentration is too high, appropriately reduce their concentrations.

3. If excessive enzyme is used, decrease the amount of enzyme.

4. If Mg²⁺ concentration is too high, reduce the magnesium ion concentration.

5. If the annealing temperature is too low, increase it appropriately or use a two-step temperature method (e.g., 94°C for denaturation and ~65°C for annealing/extension).

6. Excessive PCR cycles not only reduce amplification efficiency but also increase misincorporation rates, so the number of cycles should be reduced.

Q4: What strategies can be used to improve PCR specificity?

Answer:

Four Strategies:

1. Nested PCR (Nest-PCR): Increases sensitivity for rare target sequences; reduces the likelihood of amplifying multiple target sites; improves PCR specificity.

2. Touchdown PCR: The first few cycles use stringent annealing conditions to enhance specificity. Cycling begins at an annealing temperature approximately 5°C higher than the estimated Tm, then decreases by 1–2°C per cycle until the annealing temperature is 5°C below the Tm. Suitable for AFLP, DNA fingerprinting, etc.

3. Hot Start PCR: Delays DNA synthesis by inhibiting an essential component until the PCR machine reaches the denaturation temperature. For example, preparing the PCR reaction mix on ice to suppress Taq polymerase activity, then placing it in a preheated PCR machine.

4. Using PCR Enhancers: Reagents such as formamide, DMSO, glycerol, and betaine can lower the melting temperature, facilitate primer annealing, and assist DNA polymerase in extending through secondary structure regions. However, the concentration of enhancers should be appropriate.

Q5: What are the general principles for PCR primer design?

Answer：

1. Primer Length: 15-30 bp, typically around 20 bp.

2. Primer Base Composition: The G+C content should ideally be 40-60%. Too little G+C may result in poor amplification, while excessive G+C can lead to nonspecific bands. The GC content of the forward and reverse primers should not differ significantly. The A/T/C/G bases should be randomly distributed, avoiding stretches of more than five purine or pyrimidine nucleotides in a row.

3. Primer Structure: Avoid complementary sequences and secondary structures at the 3’ end of the primer. The bases at the 3’ end, especially the last and penultimate bases, must strictly pair correctly.

4. Restriction Enzyme Sites: These are generally added to the 5’ end of the primer. Suitable restriction sites facilitate subsequent restriction analysis or molecular cloning in experiments.

5. Primer Concentration: Each primer concentration should be 0.1-1 μmol or 10-100 pmol. The optimal concentration is the minimum amount required to produce the desired result. Excessive primer concentration may cause mismatches and nonspecific amplification, as well as increase the likelihood of primer-dimer formation.

Q6: What are the optimal conditions for cloning PCR products?

Answer:

The optimal ratio of the insert to the vector needs to be determined experimentally, with a 1:1 ratio generally being the best, though molar ratios of 1:8 or 8:1 are also acceptable. For the ligation, use 5 μL of 2X ligation buffer, 50 ng of plasmid DNA, 1 Weiss unit of T4 DNA ligase, and the insert in a total volume of 10 μL. Incubate at room temperature for 1 hour or at 4°C overnight (which can improve ligation efficiency). At both temperatures, vectors lacking T-overhangs may self-ligate, resulting in blue colonies.

Q7: Does the PCR product need to be gel-purified?

Answer:

If gel analysis shows only a single band in the PCR product, gel purification is unnecessary. However, if there is a significant amount of primer dimers, gel purification should be performed prior to cloning.

Q8: If the target fragment is not recovered, what control experiments should be performed?

Answer:

1. Plate the untransformed competent cells. If colonies grow, it indicates that the ampicillin has lost effectiveness or there is contamination by ampicillin-resistant bacteria.

2.  Transform the intact plasmid, count the number of grown colonies, and determine the transformation efficiency.  Transformation efficiency = (Total number of colonies formed) / (Total amount of DNA plated).  The total amount of DNA plated is the amount used in the transformation reaction divided by the dilution factor.For example:1 μL of plasmid (1 μg/μL) is used to transform 100 μL of competent cells.1 μL of the transformed competent cells is then diluted into 1000 μL (containing 10 ng DNA), and 100 μL is plated (containing 1 ng DNA).After overnight incubation, 1000 colonies are obtained.Transformation efficiency = (1000 colonies × 10³ ng) / 1 ng DNA plated = 10⁶ cfu/μg.If the efficiency is below 10⁸ cfu/μg, the transformation efficiency is low, and the cell transformation should be repeated.

3. If using pGEM-T as a positive control results in more than 20-40 blue colonies, it suggests that the vector has lost the T-overhang.  This may be due to nuclease contamination in the ligase, and the nuclease should be replaced.

Q9: The control experiment worked well, but the target fragment was not recovered. What could be the issue with the experiment?

Answer:

1. The target fragment is unsuitable for ligation. Gel-purified DNA fragments may develop pyrimidine dimers if overexposed to UV light, hindering ligation. The DNA must be repurified.

2. If the DNA polymerase in the PCR system has proofreading activity, the amplified product will lack the 3′-A overhang (required for cloning into pGEM-T vectors). Replace the enzyme with Taq DNA polymerase.

3. Highly repetitive sequences may be unstable, leading to deletions and rearrangements during PCR amplification. If the target fragment frequently exhibits such issues, use a recombination-deficient E. coli strain (e.g., SURE cells).

Original link：https://zhuanlan.zhihu.com/p/78193892