Recommendations for avoiding contamination during PCR
Over 10 million copies of DNA template are processed during PCR. Therefore, it is important to prevent the possibility of contamination with other templates and amplicons that are present in
laboratory. Here are general recommendations for reducing the risk of contamination:
- Preparation of DNA samples, preparation of reaction solutions, amplification and analysis of PCR products should be carried out in different territorial areas.
- Prepare reaction solutions in PCR laminar flow cabinet equipped with UV lamp.
- Use new pair of gloves when purifying DNA and preparing mixtures and solutions.
- Use reagents designed specifically for PCR. Use pipette tips with integrated aerosol filter when preparing DNA samples and reaction solutions.
- For verification of the absence of contamination, prepare a mixture sample without DNA template (negative control).
Recommendations for primer selection
For design of primers and probe, we recommend using Oligo software http://www.oligo.net/ and its analogs. For selection of oligonucleotides, follow the basic principles:
- Primer length:18-22 bp.
- Difference in melting temperatures (Тm) of the two primers shouldn't exceed 3 °С.
- Tm of primers for TaqMan PCR should be ≥ 60 °С.
- GC composition of primers should be within the range of 40 to 60%.
- Product length: 70 – 150 bp.
- Minimize secondary structures, avoid them, if possible.
- Check your primers using BLAST.
- Probe length 22-26 bp;
- Melting temperature: 68-70 °С.
- Minimum of the same nucleotides in a row (especially G: not more than 4 in a row).
- Chose DNA strand that has more C nucleotides than G nucleotides in it.
- There should be no G at 5'-end.
- Avoid self-complementarity and formation of dimers between probe and primers.
- Purity and integrity of DNA is extremely important for successful PCR. For isolation of DNA, apply conventional methods that allow further amplification of the sample.
- Avoid using PCR inhibitors (phenol, hemin, etc.) when working with the samples. In case of using gel purification, minimize UV exposure in order to prevent formation of pyrimidine dimers.
- Prepare reaction solution in a clean area, use pipette tips with integrated filter in order to reduce contamination risk.
- Optimum amount of DNA per reaction depends on the type of sample and its purity: phage lambda DNA ~0.1 ng; E.Coli DNA ~10 ng; human DNA ~10 – 50 ng.
Characteristics of amplification steps
- Initial DNA denaturation and enzyme activation
- It is very important to achieve complete denaturation of DNA template at the beginning of PCR which provides its efficient use in the first amplification cycle. If GC composition of the
is 50% or less, initial denaturation at 95 °С for 5 min will be enough.
Standard time of denaturation per cycle for real-time PCR is considered to be 15 - 30 sec at 95 °C.
Primer annealing and elongation
For TaqMan real-time PCR, annealing and elongation stages are usually combined into one step at 58-60 ºС for 60 sec.
Number of cycles for PCR Master Mix
If there is less than 10 copies of DNA template available per reaction, then efficient amplification requires not less than 40 cycles. A total of 25 – 35 cycles is enough for higher amount of