PCR success can vary depending on your experimental design.
Small errors can often result in low yields or false negative/positive products.
4 essential tips to ensure the success of your PCR reaction:
1. Primer Design
– Find the optimal primer concentration.
– To avoid secondary structure formation, a primer 18-30 nucleotides in length and a GC content of 40-60% is recommended.
– Check primer homology. Any binding between your primers or internally will result in reduced PCR efficiency.
– The binding degrees of the primers are calculated with the formula 2 (A+T)+4 (G+C).
– Finish with a G or C. Closing the 3′ end of your primary knee with a G or C will strengthen the primary extension at the extension site.
2. Target Sequence
– A PCR reaction can be affected by both the quality and quantity of your DNA template.
– A quality DNA sequence will increase the specificity of the reaction and product yield.
– Be careful to avoid contamination! Protein or chemical contamination can cause non-specific binding or completely inhibit PCR.
– Check that the 260nm/280nm ratio of your DNA absorbance is ≥1.8.
3. Reaction Reagents
– Hazardous reagents affect the efficiency of your PCR reaction. Although many laboratories use commercial off-the-shelf components, the components to know and consider are:
DNA Polymerase, dNTP, magnesium concentration.
4. Thermal Profile
– Thermocycler protocols and conditions are highly standardized for PCR. Here are three main things to consider:
” Modified PCR conditions:
” Bonding Temperature: it would be optimal to set it 3 °C below the melting temperature of your primary sequence. For further optimization the anneling temperature can be increased in steps of 1-2 °C.
” Extension time: It is recommended to set an extension time of 1 minute for every 1 kb amplicon. Optimal extension rates may depend on the processability of the DNA polymerase.