1. Except for bacterial cells, please attach DNA electrophoresis pictures for other samples and indicate the amount of sample in the picture.
2. Except for Primer (T7、T3、SP6、M13F、M13R etc.), if you design primer yourself, please indicate the name and concentration (10 pmole/µl), and provide at least 5 µl for each reaction.
3. Plasmid DNA or PCR product is dissolved in ddH2O, do not dissolve in TE Buffer to avoid interference.
4. For delivery of Plasmid DNA, the volume must be >10 µl and the concentration must be >40 ng/µl.
5. For self-purified PCR products, the volume after purification must be >10 µl, and the concentration must be >20 ng/µl.
6. After DNA sequencing is completed, the remaining DNA will be stored for one week and will be destroyed after the expiry date.

1. Residual alcohol, salt, EDTA, detergent, and organic chemicals may interfere with the Big Dye reaction.
2. If plasmid extraction is performed using conventional methods or if the plasmid has not been treated with RNase, please specify on the order form.
3. DNA should only be dissolved in pure water and should not be dissolved in buffers containing EDTA (such as TE buffer).

1. PCR products must exhibit a single band and should not appear as a smear.
2. Prior to sequencing, PCR products must undergo a purification step to remove primers and dNTPs.
3. Only one primer should be added.
4. When gel-eluting PCR products, avoid using short-wave UV exposure.

Based on the size of the DNA, the recommended quantities for a single sequencing reaction are as follows :

Confirmation is performed by running electrophoresis before proceeding with the sequencing reaction.

Confirmation is performed by measuring the absorbance values before proceeding with the sequencing reaction.

Each sequencing reaction requires 1 µl of primer at a concentration of 3.2 µM. You can estimate based on the number of sequencing reactions you plan to perform. Additionally, considering the possibility of redoing reactions, it is advisable to overestimate.
Please assist in indicating the primer concentration and volume, for example: 10 µM (10 µl). The laboratory will then dilute it to 3.2 µM before conducting the sequencing.

1. Primer length should be between 18 bp and 25 bp.

2. The annealing temperature for primers is approximately 5°C lower than the Tm value. For sequencing, the annealing temperature is set at 50°C; therefore, it is recommended to design primers with Tm values ranging between 50-58°C.

3. G-C content should fall within the range of 30-80%.

4. Avoid self-complementarity between primers or the formation of secondary structures within the primers.

5. Avoid repetitive sequences of the same nucleotide, especially sequences with four or more consecutive G nucleotides.

Conc. (pmol/μL or μM) = (A260 x100) / (1.54x nA+0.75x nC+1.17x nG+0.92x nT)。

You can download some free image viewing software online, such as Chromas, SnapGene Viewer, FinchTV.
Chromas | Technelysium Pty Ltd
SnapGene Viewer | snapgene-viewer
FinchTV | Digital World Biolog

★Machine：Applied Biosystems 3730xl DNA Analyzer

★Reagent：BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA)

★PCR System，
1. 98℃ for 5 mins，1 cycle.
2. 96℃ for 10 seconds.
3. 50℃ for 5 seconds.
4. 60℃ for 4 mins.
Repeat the steps (2. To 4.) for 26~30 cycles.
5. 4℃ for ∞，1 cycle.

Fluorescent PCR products can be used as templates for sequencing reactions, but primers with fluorescent labels cannot be used for sequencing.

The typical cultivation conditions involve using 2× LB Broth (3~6 ml) at 37°C for 6-12 hours. Additionally, commonly used antibiotics include Ampicillin, Kanamycin, Chloramphenicol, Tetracycline, Spectinomycin, and Zeocin.

If your bacteria do not thrive under the mentioned conditions or if you are using antibiotics not listed above, please specify.

★ Our laboratory currently provides a list of commonly used sequencing primers (please refer to the Primer list for Sanger sequencing services). If you are unable to confirm or select from the list, please provide the full name and brand of the vector used on the order form. Additionally, indicate whether you want sequencing from the Forward end, Reverse end, or both. Our laboratory will confirm and select the appropriate primers for you.

★ If you require a Universal Primer not listed in the table, please contact our laboratory by phone for further assistance.