Genetic Services

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.

If gel purification is not performed and only a purification kit is used, it often leads to double peaks or messy peaks in the sequencing results. This is mainly caused by non-specific amplification products or incomplete removal of the original PCR primers. Most so-called PCR "purification kits" primarily recover the product but do not effectively purify it. They certainly cannot remove non-specific amplification products, and often they do not completely remove all PCR primers. These residual primers can participate in the sequencing reaction, causing messy peaks.

Firstly, fresh bacterial culture is easy to grow, allowing for more DNA yield, while also maximizing the purity of the strain. If providing fresh culture, seal the cap with parafilm to prevent leakage. Alternatively, pellet 4–5 mL of the cultured bacteria, pour off the supernatant for easier shipping. When mailing, it's best to use a box to prevent the tube from being crushed during transit.

The PCR product must first be run on an Agarose gel. The target band is excised and then purified using a gel extraction kit. The eluted product should be dissolved in ddH₂O.

Generally, bacterial forms include: plate cultures, stab cultures, glycerol stocks, or fresh liquid cultures. We recommend sending stab cultures or fresh liquid cultures.Plate cultures are particularly inconvenient for shipping; we often receive plate cultures where the petri dish has broken during transit, rendering the sample unusable and requiring the client to resend. This wastes time and the client's sample. If it is a very important sample for the client, the consequences are even more significant. Glycerol stocks are prone to contamination.
When preparing stab cultures, add agar medium to a 1.5 ml tube, and stab the bacteria into the agar medium (solid) using a toothpick. Incubate at 37°C overnight, and it's ready. Stab cultures can be stored at 4°C for several months, are less prone to contamination, and are easy to ship.