Etiket Arşivleri: PCR

Every PCR reaction has the same problem:

Very little amplification of target DNA, but more amplification of non-target DNA. However, you can solve the problem of your PCR reactions by using some additional reagents. It is up to you to determine which PCR reagent does what and which condition is best for you.

PCR extension reagents generally work in one of the following two ways:

① By reducing secondary structures and increasing amplification of target DNA ↑

② By reducing non-specific binding and decreasing amplification of off-target DNAs ↓

Mg⁺² is required for polymerase activity. Withoutsufficient Mg⁺², taq polymeraseremainsinactive.The correctconcentration of Mg⁺² increasesspecificity. LowMg⁺² concentration increasesnon-specific binding.

Bovine serum albumin is a commonly used addition in various molecular biology applications, especially in restriction enzyme digests and PCR. In PCR, BSA can help combat contaminants such as phenolic compounds. It is also reported to prevent reaction components from sticking to tube walls.

Improves amplification of GC-rich and difficult DNA targets. Ideal for use on sequences known to be difficult to amplify. Reduces DNA Tm temperature.

The use of chemical components such as Triton X-100, Tween 20, NP-40 is also thought to reduce secondary structures. The use of these reagents may increase PCR efficiency but may also increase non-specific amplifications. Therefore, they should be used with caution.

It is thought to reduce secondary DNA structures. Therefore, it may be advisable to add GC-rich constructs during PCR analysis. However, DMSO can also cause a decrease in Taq polymerase activity, so it is very important to balance the two.

It is an organic PCR extension reagent. It works to clean up dirty PCR reactions by reducing non-specific binding.

Although all these components improve PCR results, it is not possible to predict at the first stage what is best for your analysis. It takes a lot of experimentation to optimize a PCR reaction.

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.