Large-Scale DNA Sequencing Services
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 | Q: How should I send my sequencing samples? |
| | A: To submit samples, please download and complete Sequencing Order Form and send it to gene@genscript.com. Please also attach a completed hard copy to your samples and send them to the address specified on the form. |
| Q: How should I determine the concentration of my samples? |
| | A: Please measure the OD with a spectrophotometer at 260 nm and 280 nm. The OD 260 values should be in the range of 0.05 to 0.8 to give reproducible and reliable results. The OD 260/280 ratio should be between 1.8-2.0. Values lower than 1.6 and higher than 2.0 indicate contaminants in the sample that interfere with the determination of the concentration and would almost certainly inhibit the sequencing reaction. If possible, we recommend measuring the ODs at 230 nm and 320 nm, too. A high value at OD 320 indicates a contaminant. These should be ideally 0.0. The OD 230/260 ratio should be lower than 0.6. To be on the safe side, we recommend ensuring the quality of your DNA by an agarose gel, too. |
| Q: Which method for purification should I use? |
| | A: For plasmids, we recommend using commercially available plasmid mini scale preparation kits employing spin columns. From our experience, repeating the washing step will improve the quality of the DNA and therefore improve the sequencing results. Please do not send DNA prepared with alkaline lysis methods or with the boiling method without column purification. For PCR products please use commercially available PCR purification spin column kits. |
| Q: Can I send my DNA samples in Tris-EDTA (TE)? |
| | A: No, please do not. EDTA binds bivalent cations such as Mg2+ that are essential for the Taq polymerase. Your DNA is stable in double distilled water or Tris-HCl at room temperature for several days or even longer if it is dried down. |
| Q: Should I submit my DNA samples cooled? |
| | A: This is not necessary, DNA is stable in water at room temperature for several days. Just send your samples either in Tris-HCl, distilled water or air dried. Please, do not use EDTA as this will inhibit the Taq polymerase. |
| Q: Can I submit a few of samples to GenScript for sequencing? |
| | A: GenScript only provide the large-scale sequencing services, which currently accepts orders of 192 samples or more. |
| Q: What was required for sequencing samples? |
| | A: Our requirements for sample submission are listed in the table below:
| Template | Concentration | Volume/Reaction | Solvent |
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| Genomic DNA | 50-100 ng/μl | ≥5 μl | ddH2O or 10 mM Tris (without EDTA) | | PCR Fragments | 20 ng DNA/100 bp | ≥8 ul | | Plasmid DNA | 200 - 500 ng/reaction | ≥8 μl |
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| Q: Which E. coli strain should I use? |
| | A: Successful isolation of plasmid DNA is possible from most E. coli strains, though the strain which is used can have a significant influence on the quality of the purified DNA. Host strains such as DH10b, DH5 alpha, XL10 Gold and TOP10 normally yield high quality DNA in combination with many commercially available plasmid DNA isolation kits and are therefore ideal for the propagation of plasmids to be sequenced. Lower quality DNA is derived from strains producing large amounts of carbohydrates, which are released during lysis and inhibit enzyme activity. Examples are HB101 and its derivatives such as TG1 and the JM100 series. Also strains with medium or high levels of endonuclease activity like HB101, JM101 or BL21 generate DNA of lower quality, hence a proteinase K treatment should be considered. |
| Q: Do I need to clean up my PCR product if there is only one band? |
| | A: Primers and dNTPs must be removed from the product. The primers would produce a mixed sequence (5% contamination with a primer results in unreadable mixed sequences!) and the dNTPs would interfere with the dNTP/ddNTP concentration in the sequencing reaction. We recommend purification by using commercially available PCR spin column kits. |
| Q: I see unspecific bands in my PCR reaction. Do I need to gel purify my product? |
| | A: If you want to employ one of the PCR primers in the sequencing reaction you must elute the product of interest from an agarose gel. Otherwise, a mixed sequence will be produced as the primer is most likely to bind both the specific and unspecific products. If you employ a specific nested primer in the sequencing reaction, it might work. However, producing only one product in the PCR reaction is the best guarantee for a good sequence. In almost all cases, improving the PCR conditions (specific primer design, higher annealing temperature and/or lower primer concentration) helps to avoid unspecific products. |
| Q: What is the guaranteed read length each reaction? Is there a free trial? |
| | A: GenScript can guarantee the read length of 800 bp. We offer a free trial of one sequencing run, with typical readout of about 800 base. |
| Q: What is the average success rate of GenScript DNA sequencing reaction? |
| | A: We have observed in large scale trial with PCR templates an average success rate of greater than 90%. Other trials of GenScript under less stringent production condition (microbial genome sequencing) with templates of highly variable quality the success rate was approximately 80%. |
| Q: Are there some templates that are harder than others to sequence well? If so, what are they, and why? |
| | A: We implement robust sequencing chemistry to guarantee the sequencing accuracy and coverage of 99.0%, however, some templates are not able to generate good sequence data:
High G/C templates (>70% GC) may not stay denatured during the synthesis cycle. This makes it difficult for polymerase to add dNTPs to the growing sequence ladder; High AT templates (>70% AT) may form into secondary structures that causes poor polymerase binding to the template and sequencing reads; Thawed DNA Templates, which may become degraded by repeated frozen and thaw, leading to much shorter sequencing reads and poor quality data; polyA sequences, which can be difficult for BigDye Terminator chemistry to overcome.
Please remember that if any difficulties arise, we will do our very best to get you the data that you need. For the best sequencing results, please handle your samples according to our recommendations, and call us if you have any questions regarding our services. Other anomalies do exist and we will try to keep you informed if any may appear in your samples. |
