The results of PCR product electrophoresis are only a rough qualitative result. Non-specific PCR amplification products that differ in size by only a few bases from the target fragment cannot be distinguished by the naked eye. However, Sanger DNA sequencing reactions are sensitive and objective, and can directly reflect the template itself. If you are not sure about the purity of the PCR product, we hope you can clone the PCR product to ensure good sequencing results.
PCR primers can be used as Sanger Sequencing primers, but the longer the PCR primer, the less satisfactory the sequencing result may be, therefore, not all PCR primers are suitable to be used as sequencing primers.
(1) Continue sequencing using reverse primers to correct the deletion site and achieve a pass-through. Alternatively, clone the PCR product into a plasmid and pick a single clone for sequencing.
(2) If it can be determined that there should not be a missing site in this PCR fragment, then the PCR reaction conditions can be changed and re-amplified.
Take polyT as an example, after polyA/T structure, there are often shifts, double peaks and sets of peaks, while after polyG/C it will often lead to attenuation of sequencing signal or direct interruption.
In the case of confirming the primer, plasmid extraction concentration, reaction arrangement and other conditions are fine, the sequencing result with a messy peak pattern and signal value less than 100; then the result will be judged as no signal for sequencing.
(1) excessive amount of template in the sequencing PCR reaction
(2) degradation of PCR products
(3) Contaminants in the sample that inhibit DNA polymerase activity
(1) reduce the amount of template in the sequencing PCR reaction
(2) storage of PCR products at 4 °C for no more than 48 hours, long-term storage at -20 °C is required
(3) Repurify the sample
(1) re-capillary electrophoresis by dilution of the upload product with HIDI
(2) set a shorter injection time
(3) resequencing the PCR and reducing the amount of template in the sequencing reaction
(1) capillary micro-blockage
(2) presence of impurities in the capillary electrophoresis process
(3) If using BigDye XTerminator purification kit, microbeads in XTerminator solution enter capillary electrophoresis together with the product, resulting in abnormal feeding
(4) POP gel, cathode buffer exceeding the length of use
(5) The amount of sample entering capillary electrophoresis is too much
(1) rinse the capillary tube with Fill array and re-capillary electrophoresis
(2) rinse the gel pump and tube with washing solution and then rerun capillary electrophoresis
(3) re-capillary electrophoresis after BDX shaking and sufficient centrifugation to allow the microbeads to precipitate
(4) replace new POP gel, cathode buffer
(5) Re-capillary electrophoresis after diluting the upper product with HIDI or reducing the injection time
(1) signal drop before sequence end A-peak ending: contains non-specific amplified sequence with strong signal and weak signal of target sequence;
(2) severe degradation of the product;
(3) DNA polymerase extension efficiency is reduced due to encountering complex regions.
(1) re-extract nucleic acids with increased sample size and re-assay
(2) resequencing PCR and purification
(3) redesign new primers or reverse sequencing
For Research Use Only. Not for use in diagnostic procedures.