1. How can I design suitable primers?

Answer: First, we need to know the sequence of the target gene. Once we have the sequence, we can use Oligo or Primer software to design the primers. When designing primers, we must pay attention to factors such as primer length, GC content, and Tm value.

2. Why are there no bands in the PCR product?

A: There are several possible causes. (1) Primer-related issues: Primers may have degraded due to improper storage or may have been designed poorly. Check the primer sequences and resynthesize the primers; if previous experiments were successful, this possibility can be ruled out. (2) Template-related issues: Prolonged storage or repeated freeze-thaw cycles of the template can lead to degradation; If the template DNA is derived from plants, fresh leaves from plants that are not rich in polysaccharides or polyphenols should be selected. When extracting DNA or RNA, the material should be ground using liquid nitrogen, and the process should be performed quickly; if the template is cDNA, the concentration, purity, and integrity of the RNA must be assessed; (3) Enzymes: Enzyme inactivation; try using a new enzyme or a different type of enzyme for the experiment; (4) Reaction system: The reaction system is inappropriate; (5) Reaction protocol: Are the pre-denaturation or denaturation temperatures too high, causing enzyme inactivation? Or is the denaturation temperature too low, preventing the template strands from unwinding? If these causes are ruled out, the annealing temperature may be inappropriate, and a temperature gradient should be set to optimize the conditions through trial and error.

3. Non-specific amplification occurs?

Answer: (1) Self-hybridization of the primers leads to the formation of secondary hairpin structures; (2) Primers hybridize with each other instead of binding to the DNA template, resulting in primer dimers; (3) The template DNA is contaminated or there is excess template; (4) The reaction protocol is not optimized: If bands larger than the target band appear, appropriately shorten the extension time or reduce the number of cycles; conversely, extend the extension time and increase the number of cycles.

4. Why do PCR products show tailing during validation gel electrophoresis?

Answer: (1) The agarose gel powder did not dissolve completely during gel preparation; (2) Excessive nonspecific amplification; (3) Excessive enzyme concentration; (4) If crescent-shaped bands appear, this indicates that the voltage was too high.

5. Why are there bands in the blank control?

Answer: (1) The primer design is inappropriate, resulting in the amplification of non-target gene fragments; (2) If the band in the blank control is the same size as the target band, this indicates contamination. Replace the buffer, ddH₂O, or primers and repeat the experiment; (3) If the intensity of the band in the blank control is weaker than that of the target band, reduce the number of cycles to prevent the blank control from amplifying the target band.