Due to confidentiality reasons, we retain sequencing samples for only one month. So, if you want to run another sequencing reaction, please do so promptly; otherwise, you will need to submit a new sample.

Due to complex structures like hairpins and palindromes of unknown origin, there may be sudden signal weakening or disappearance. Sometimes, sequencing cannot proceed at all. While the DNA base arrangement shows no specific abnormalities, it could be due to the overall occurrence of complex structures, hindering the reaction.

PCR products below 150 bp are challenging to purify and quantify. For sequencing purposes, PCR products should generally have a length greater than 150 bp. For a 150 bp PCR product used for sequencing, considering the removal of primer sequences (approximately 40 to 50 bp) and some unreadable bases at the sequencing start, the actual useful sequence obtained is only a few dozen bases. This is under the most ideal assumptions, and the likelihood of sequencing failure for such short fragments is significant. Therefore, cloning is typically required before sequencing.

Due to the continuous G/C structures, sometimes the reaction cannot proceed normally. If the structure is near the downstream of the primer, there might be only a short segment of signal in the reaction, and sometimes there may be no signal at all. For sequencing, which already has inherent uncertainties, such situations pose a significant risk of failure.

For host bacteria, it is recommended to use DH5α, DH1, and C600 E. coli strains. XL1-Blue E. coli strain is also suitable, but it grows relatively slow. E. coli strains such as JM series, TG1 series, and HB101 series should be avoided as they produce a large amount of carbohydrates (i.e., sugars). Other host bacteria may potentially affect sequencing results.

Not all primers suitable for PCR reactions are suitable for sequencing. Sequencing primers have higher requirements, they must perfectly match the template (occasionally allowing a few degenerate bases at the 5' end), have a length of around 20 bases, moderate GC content, and must be sufficiently pure for sequencing

This situation is usually characterized by peak overlapping in the sequencing results after consecutive A or T structures, and it is an intrinsic issue with the sample. We should report such issues to the customer as with normal sequencing, and the reaction will be charged at the regular rate.

The sequencing results show overlapping peaks, the sequencing reaction signals are good, but the sequencing results are chaotic, meaning there are two peaks at the same position. Possible reasons include: the sample is not a single template, there are impurities in the PCR product, the plasmid is a double clone product, and the primer specificity is not high, with two binding points. Although not visible during PCR product electrophoresis, the sequencing results clearly illustrate this issue.

The obtained results do not align with your expected direction, which may be due to the non-directional insertion of the fragment, a common occurrence in T/A cloning of PCR products. Sometimes, if the sequencing primer requested by the customer is close to the cloning site and the reverse primer is relatively far from the cloning site, for better results, we may choose to sequence with the reverse primer. In such cases, using visualization software like Chromas allows you to reverse the sequence and obtain the forward sequence.

Common forms of bacteria include bacterial liquid, plate-cultured bacteria, stab-cultured bacteria, glycerol-preserved bacteria, etc. We recommend customers to submit samples in a form that is convenient and less prone to contamination, with stab-cultured bacteria being preferred. For customers in Shanghai, 2 ml of overnight cultured bacterial liquid is recommended.

Due to EDTA being a potential inhibitor of Taq polymerase, and DNA sequencing reactions involving the polymerization reaction of Taq enzyme, it requires an optimal enzyme reaction condition. Therefore, when dissolving DNA sequencing samples, it is recommended to use sterile water for dissolution to avoid potential interference.

If only a single signal appears at a position where you expect a heterozygous signal, it may be because the proportion of your sample's mutated template to the normal template is not at a detectable concentration. The signal strength of the sequencing reaction is directly related to the amount of template, and if the proportion of the mutated template is very low, the instrument may automatically consider it as background signal, making it difficult to detect. Only when the amounts of normal and mutated templates in the sequencing reaction system are relatively close can the presence of th…

There are two possible reasons for sequence variations when cloning PCR products into vectors for sequencing: Firstly, mismatches may occur during the PCR amplification process. Additionally, mutations can potentially arise when cloning the fragment into the vector. Secondly, the accuracy of sequencing is not 100%.

There could be several reasons, including variations between individual samples and issues with the accuracy of sequencing. The analysis of sequences by automatic sequencers is not 100% accurate. It is recommended to perform at least one bidirectional sequencing to minimize sequencing errors. Although we make every effort, we cannot guarantee complete consistency with the literature sequence. However, our sequencing report reflects the authentic results of the customer's sample sequence.

The background smear in the sequence trace is caused by fluorescence dyes. If it is too strong, it can affect the sequencing results. The impact depends on the signal-to-noise ratio, and the results we provide generally have a signal-to-noise ratio of 98% or higher.

Firstly, we will verify whether the sequencing results we provided correspond to your sample number. If it matches your sample, we cannot determine whether the sequenced sequence aligns with your expected results due to a lack of information about your experimental background. Our capability is limited to checking if the sequencing results sent to you match the sample you provided."

The possible reasons are the same as mentioned earlier. The enzyme cutting site you cloned may be too close to your sequencing primer, and the initial sequence is very noisy, making it almost impossible to discern. It is possible that the cutting site may not be clearly visible or not visible at all. Usually, we try to choose primers that are farther away from the cutting site. Of course, if there are unexpected issues with the sample, such as empty vector or vector self-ligation, the cloned enzyme cutting site may also not be visible.

If you can't find the primer sequence used in the sequencing, that's normal because primers themselves are not labeled, so they won't be found in the sequencing report. If you can't find the amplification primer from the cloned fragment, it may be because the enzyme cutting site you cloned is too close to your sequencing primer, and the initial sequence is very noisy, making it almost impossible to discern. In such cases, the complementary sequence of the primer needs to be searched, especially if the insert fragment has a reverse orientation.

Due to the gradual decrease in signal intensity in the sequencing reaction, the signals at the end of the sequence are weak, resulting in a naturally noisy chromatogram. Additionally, the resolution of the sequencing gel plays a role, and if the bases are not well-separated, 'N' values may be generated. Under normal circumstances, the ABI 377 sequencer can accurately read an effective sequence of 500 bases."

This is mainly due to interference peaks caused by residual dye monomers, which overlap with the normal sequence peaks. In addition, there is a stabilization period in the voltage at the beginning of the sequencing electrophoresis, so fragments closely following the primer, typically 20-50 bp, are often unclear, and sometimes even longer."