Q&A

We use advanced synthesis techniques and strict quality control processes to ensure the accuracy of single stranded DNA sequences. In addition to basic concentration purity testing, there are also sequencing validation, enzyme digestion validation, and assembly validation.

You can increase the amount of DNA sample, or use a slightly more sensitive electrophoresis method (such as polyacrylamide gel electrophoresis).

Avoid DNA nuclease contamination and ensure DNA integrity.

Adjust the electrophoresis time and voltage to enhance the gel concentration to prevent DNA from leaving the gel.

For DNA bands with similar molecular size, increase the electrophoresis time and approve the gel concentration to improve the resolution.

Avoid high temperature heating of DNA strands before electrophoresis.

1. You need to check the purity, concentration, and integrity of the template DNA to avoid cross contamination.

2. Ensure the activity and usage of Taq enzyme, and avoid using expired enzymes.

3. Choose the correct primer concentration and sequence to avoid primer degradation.

4. Optimize the amplification program, including annealing temperature, cycle times, etc.

5. Ensure that the salt concentration and pH value of the reaction system are appropriate.

6. Use standard reaction tubes and siliconized tubes to ensure the temperature control system of the amplifier is functioning properly.

When designing PCR primers, the following key parameters need to be considered to ensure the effectiveness and specificity of the primers:

Primer length: usually 18-30 nucleotides (nt), with around 20nt being the most commonly used. The length of upstream and downstream primers should be roughly equal, with a maximum difference of no more than 3 base pairs (bp). Short primers may lead to non-specific amplification, while longer primers, although more specific, may form secondary structures within the primer.

GC content: between 40% and 60%. A low G+C ratio results in poor amplification efficiency, while a high ratio can easily lead to non-specific bands. ATGC is best distributed randomly to avoid duplicate motifs.

Melting temperature (Tm value): between 55-75 ℃, annealing temperature needs to be 5 ℃ lower than melting temperature. The difference in Tm values between primer pairs should not exceed 2-3 ℃. The Tm value affects the binding efficiency between primers and target DNA.

Primer amplification span: 200-500bp is suitable for ordinary PCR, while 70-150bp is suitable for real-time fluorescence PCR.

Secondary structure: Primer design should preferably cross introns to avoid hairpin structures. The main reason why some primers are ineffective is the influence of the DNA secondary structure in the primer repeat region.

Base distribution: The distribution of the four bases in primers should be kept random to avoid designing primers containing 5 or more consecutive identical bases, as this may lead to non-specific amplification and the formation of hairpin structures or dimers during PCR.

Complementarity: Avoid continuous 4-base complementarity between primers or primers themselves, as this may lead to non-specific amplification or affect the efficiency of PCR.

The Tm value of primers can be determined using the following formula:

For primers shorter than 20 bases: Tm=2 ℃× (A+T)+4 ℃× (G+C)

For primers with more than 20 bases: Tm=81.5+0.41 × (GC%) -600/L, where L is the length of the primer.

Primer concentration: The final concentration of primers in general PCR reactions is 0.2~1 μ mol/L. Within this range, primer concentration will not have a significant impact on the amount of PCR products. When the primer concentration is below 0.2 μ mol/L, it may lead to a decrease in product quantity; When the primer concentration is too high, it will greatly increase the probability of primer errors, leading to the synthesis of non-specific products, and also increasing the formation of primer dimers.

Enzyme concentration: While ensuring effective PCR, reducing the enzyme concentration as much as possible can improve reaction specificity. It is advisable to add 1-2.5 units of Taq DNA polymerase to every 100 μ L reaction system. The amount of enzyme used may vary depending on the template DNA or primer. For example, the amount of enzyme required when using plasmid DNA as a template is less than that required when using chromosome DNA as a template.

We recommend storing all culture media at 2-8 ℃ and using them up within a week.

PCR reactions typically involve three basic steps: denaturation, annealing, and elongation. According to the length and complexity of the target sequence, different PCR reaction step methods can be selected:

Three temperature point method: This is the standard PCR reaction step method. In the denaturation step, double stranded DNA is denatured into single stranded DNA at 90-95 ℃; In the annealing step, when the temperature drops to around or below the Tm value of the primer, the primer binds to the complementary region of the DNA template to form a hybrid chain; In the extension step, heat-resistant DNA polymerase catalyzes the extension reaction starting from the primer in the 5 '→ 3' direction.

Two temperature point method: For shorter target sequences (such as 100-300bp), the two temperature point method can be used. In this method, annealing and elongation temperatures can be combined into one, usually using higher temperatures (such as around 65 ℃) for annealing and elongation reactions. This method simplifies the PCR reaction steps and shortens the reaction time.