Sample Submission Guidelines
Sanger Sequencing Q&A
General Questions
- Why does the Sanger sequencing result say that the template is promiscuous when the electrophoresis assay is good?
- 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.
- Can PCR primers be used as Sanger Sequencing primers?
- 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.
- What is a base deletion in Sanger Sequencing?
- Base deletions are commonly found in PCR products, especially in PCR fragments amplified from the genome.
- How to use Sanger Sequencing to resolve base deletions?
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(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.
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(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.
- What are the effects of duplicate structures on Sanger Sequencing?
- Duplicate structures will lead to slippage of the sequencing replicate frame and confusion of the peak pattern after the duplicate structure.
- How to use Sanger Sequencing to resolve duplicate structures?
- Sequencing the template with reverse primers to the duplicate structure will complete the splicing of the full length of the template.
- What is the Sanger sequencing result of Poly structure?
- 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.
- What does a sequencing peak plot with a front bimodal result look like in Sanger Sequencing?
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(1) The product contains a small fragment of PCR product
(2) The primer has two binding sites and one of the results is interrupted
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(1) The product contains a small fragment of PCR product
- How to solve the front bimodal result in Sanger Sequencing?
- Reverse sequencing with a different primer.
- What should I do if there is no signal in Sanger Sequencing?
- 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.
- Why can't I find the primers in the Sanger Sequencing results?
- Sequencing results usually start to peak 30-50bp after the primers, so the primers are not found in the original results.
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What are the causes of weak sequencing signals in Sanger Sequencing?
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(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
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(1) excessive amount of template in the sequencing PCR reaction
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What if the sequencing signal is weak?
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(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
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(1) reduce the amount of template in the sequencing PCR reaction
- What are the causes of a strong sequencing signal in Sanger Sequencing?
- The amount of template in the sequencing reaction is too much, resulting in a high signal.
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What should I do if the sequencing signal is too strong?
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(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
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(1) re-capillary electrophoresis by dilution of the upload product with HIDI
- What is the reason for the delay in sequencing peak emergence in Sanger Sequencing?
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(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
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(1) capillary micro-blockage
- What should I do if there is a delay in sequencing peaks?
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(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
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(1) rinse the capillary tube with Fill array and re-capillary electrophoresis
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What are the causes of interrupted sequencing signals in Sanger Sequencing?
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(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.
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(1) signal drop before sequence end A-peak ending: contains non-specific amplified sequence with strong signal and weak signal of target sequence;
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What should I do if my sequencing signal is interrupted?
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(1) re-extract nucleic acids with increased sample size and re-assay
(2) resequencing PCR and purification
(3) redesign new primers or reverse sequencing
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(1) re-extract nucleic acids with increased sample size and re-assay
For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.