Sequencing
- Rapid & Accurate system: Results will be sent within 24 hours after receipt of samples. (Except for samples that require purification)
- High quality & Long read length : 700bp.(phred score 20)
- Customized cause analysis services
- SAS(SolGent Analysis Services): Based on a web system, Real-time monitoring “ from ordering to result check” is possible.
- Free Bioinformatics Analysis Service: Vector Screening, Blast, low-quality trimming …
DNA Sequencing is the process of determining the precise order of A, T, G, C Nucleotides within a DNA molecule. Based on Sanger method, SolGent provides standard sequencing and primer walking service.
- Analysis Kit: BigDye® Terminator v3.1 cycle sequencing Kits
- Analyzer: ABI 3730XL DNA Analyzer (50 cm capillary)
Standard sequencing
Sample Preparation
. Template Preparation
Template | Spectral properties | Volume | DNA Purity | |
Purified PCR product | size size≤200 bp 200 ~ 500 bp 500 bp≤ 1000 bp≤ | Concentration 10 ng/ul 20 ng/ul 25 ng/ul 30 ~ 60 ng/ul | 10 ul/1reaction (5 ul/1reaction required when adding reaction) | ① Electrophoresis ② Absorbance Determination |
Plasmid | ≤4 kb 4 kb≤ | 100 ~ 150 ng/ul 200 ng/ul | ||
Large DNA (BAC) | 500 ng/ul ≤ | |||
– 1 tube (well) = 1 kind of DNA, 1 tube (well) = 1 kind of primer.
– DNA elution: It is recommended to use the solution containing the sterilized third distilled water or the purification kit.
– Plasmid: Please avoid host bacterial strains such as HB101, MV1190, JM109, XL1 Blue, JM101 (JM 100 series), TG1 and TG2.
– PCR product: EXO / SAP purification is not recommended. If you have purified EXO / SAP, we recommend sequencing with internal primer.
– 96-well plate sample: It is recommended to use a strip cap to prevent cross-contamination.
. Primer Preparation
Primer | Concentration | Volume | Note |
Customer-designed primer | 5 ~ 10 pmole/ul(5 uM) | 10 ul/1 reaction | - Prepare F / R primer separately. - Primer Tm : 50 ~ 60 ℃, GC content : 40 ~ 60 %, |
SolGent’s Universal Primer | Not required | Not required |
– Reference information for creating sequencing primers:
The sequence must match(100%) between the template and the primer.
There should be no palindromic sequence in the primer sequence. (Especially 3’end of primer position)
f there are more than 4 pairs of loop-structures, the reaction often does not work. (Especially 3’ end of primer position)
Recommended Primer Tm : 50~60 ℃
Recommended Length : 18~24mer
. Analysis Progress
– Retention Period of Customer’s Sample & Primer : 1 week
– Customer’s Extension Request : Sample 1 month, Primer : 3 months
– Retention Period of Sequencing Data : 6 months
. SAS (SolGent Analysis Services)
Sequencing Online ordering system allows you to check all the progress from ordering to result check in real time. We also provide enhanced customer satisfaction services through the updates of the latest Blast D.B and support for a variety of free bioinformatics analysis services.
– SAS free bioinformatics analysis services: vector screening, BlastN/X, low-quality trimming, PDF result file
. Reanalysis & Difficult template sequencing
If the sequencing results are not good, proceed with a free reanalysis to find out the cause. One or two samples of the same result are selected first and the representative reaction is performed. In addition, we have experimented with the changed conditions and provide improved sequencing results for some difficult-to-analyze samples such as GC rich template, GT rich template, secondary structure, poly N nucleotide, and repeat structure template.
Primer walking
Primer walking is used when the size of the DNA template is too large to get a result with a single run. It is mainly used for DNA sequencing of about 2 to 15kb. For example, if you are sequencing about 3kb DNA inserted in a vector, sequencing will proceed with a vector universal primer F / R. Then, design a new inner primer based on the sequencing results and sequence the inner part that was not read in the first sequencing. This process is repeated, complete a 3kb DNA sequence. SolGent provides full service for inner primer design, synthesis, sequencing and sequence alignment.
