Gene Synthesis Service

Overlapping peaks (mixed sequences) in PCR product sequencing generally occur for the following reasons:
(1) The PCR template is impure or the PCR primers lack specificity, resulting in amplification products that include fragments of similar length to the target fragment, which cannot be separated even by gel electrophoresis. Sequencing such PCR products results in overlapping peaks.
(2) Structural reasons can cause overlapping peaks in PCR product sequencing. The presence of polyA/G/C/T tracts or other complex structures of unknown origin can lead to overlapping sequencing results.

We use horizontal agarose gel electrophoresis, adding 0.5 µg/mL EB to the gel, and include a standard sample of known concentration. After electrophoresis, compare the brightness under UV light to judge concentration and purity. This method more directly and accurately assesses whether the sample contains genomic DNA, RNA, etc., and can also distinguish different conformations of the extracted plasmid DNA.
The three conformations of plasmid DNA refer to: during the plasmid extraction process, due to various factors, the supercoiled covalently closed circular (CCC) plasmid can have one strand break, becoming open circular (OC). If both strands break, it becomes linear (L). These three forms have different migration rates. Typically, supercoiled (SC/CCC) migrates fastest, followed by linear (L), and open circular (OC) migrates slowest.
Using a UV spectrophotometer or the EB-standard DNA comparison method only detects the concentration of the extracted product. However, due to potential interference from RNA, protein, genomic DNA, etc., in the extracted plasmid DNA, the concentration value obtained may be meaningless.

General requirements for sequencing plasmid DNA:
(1) High DNA purity (A260/A280 ratio 1.6–2.0). It must not contain mixed templates, RNA, genomic DNA, or proteins.
(2) Dissolved in ddH₂O. The solution must not contain impurities such as salts or chelators like EDTA, as these will interfere with the sequencing reaction.

(1) The amplification product must be specific, with a single band. If non-specific amplification products are present, it is generally difficult to obtain good sequencing results.
(2) Gel extraction and purification must be performed.
(3) DNA purity (A260/A280) should be between 1.6–2.0, with a concentration ≥ 50 ng/µL.