Q1: The amplification efficiency is low, and there are no amplification bands in the experimental group.
A1: (1) Primers
Check if the artificially synthesized primers have degraded due to improper storage conditions; Whether the primer design is reasonable, BLAST can be used to check primer specificity or redesign primers.
(2) Template
Long term storage and repeated freezing and thawing can lead to template breakage, ring opening, or degradation. Freshly prepared DNA double strands should be used as templates; If the template GC content is too high, it will cause the double stranded DNA to be unable to open. At this time, adding PCR Enhancer (item number P021) can effectively reduce the unwinding temperature; If the template is cDNA, it is necessary to confirm the purity and integrity of the RNA used for reverse transcription.
(3) Enzymes
The enzymes used in the reaction are inactivated due to improper storage or transportation. It is recommended to replace them with new enzymes or re experiment with enzymes from another source.
(4) Amplification system
The reaction system configuration is incorrect, it is recommended to repeat the experiment.
(5) Reaction program
Check if the denaturation temperature is accurate and if the PCR instrument indicates a temperature that matches the actual temperature. If the temperature is too high, the enzyme will quickly become inactive in the first few cycles, while if the temperature is too low, the template denaturation will not be complete; The annealing temperature is not suitable. A gradient can be set for the annealing temperature to explore a suitable annealing temperature; Set annealing temperature based on primer Tm value, such as primer Tm value≥ At 72 ℃, the annealing step can be removed and the subsequent extension step (two-step PCR) can be carried out directly.
Q2: The brightness of the amplified bands is not very bright.
A2: (1) Primers
Check if the artificially synthesized primers have degraded.
(2) Template
Firstly, confirm the quality of the template. Long term storage and repeated freeze-thaw cycles can cause template breakage, ring opening, or degradation. Freshly prepared DNA double strands should be used as the template; If the template is not available, the first amplification product can be diluted in multiples and used as a template for secondary amplification.
(3) Reaction program
You can try using the Touch Down program; Extend the extension time and increase the number of cycles.
