Sanger Sequencing

Sanger Sequencing is the chain termination method for determining the sequence of nucleotide bases in a piece of plasmid DNA or PCR product. 


Features:


No stipulation in minimum sample size for shipping

Read length: 800 bp

Result delivery within 3-5 working days



Single Pass DNA Sequencing

Customer provides purified PCR products or purified plasmids before submission.


Ready-to-Load Sequencing (We accept BDT V.3.1)

Customer performs 3 steps which are cycle sequencing reaction, post-sequencing clean-up, and dried cycle sequencing products steps. And your dried sequencing products should be wrapped with aluminium foil before submission. ATGC will dissolve the dried cycle sequencing products  and denature before load into Genetic Analyzers. 

We accept sequencing reactions done by ABI® BigDye Terminator Cycle Sequencing Kit v3.1. 


Ready-to-Load Sequencing with clean-up (We accept BDT V.3.1)

Customer performs cycle sequencing reactions and send us in single tubes or 96-well plate which are wrapped with aluminium foil. ATGC will perform the post sequencing clean-up step to eliminate excess dye terminators before analysis on our Genetic Analyzers. 

We accept sequencing reactions done by ABI® BigDye Terminator Cycle Sequencing Kit v3.1. 


Difficult Template Sequencing

Refer to template with high GC-rich/ AT-rich/ GT-rich/ repetitive sequence

The recommended concentration and volume of sample and primer as this our guideline in table : 



The most important factors to success of the Sanger DNA sequencing reaction are the purity and sufficient amount of DNA template with the presence of the specific sequencing priming site. ATGC company performs the suitable DNA quality and quantification by agarose gel photos. We generate various patterns of DNA templates band to guide our customers in using agarose gel to make an approximate estimation of whether the amount of DNA template has enough to directly proceed with cycle sequencing.

Guides on preparations and requirements for purified DNA samples as this our guideline in picture and table below








The Criteria for obtaining low volume samples of Unpurified PCR Product as show in picture and table below :






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As picture and table, show the different quality of the samples depending on the conditions in the PCR reaction of each laboratory. A good sample quality should have a clear band and a single band which can indicate that the sample is sufficient and free of impurities. It is suitable to be used for purification to remove excess or irrelevant parts from the PCR process. 

In the case of sample that expresses multiple bands or other unrelated bands are found, the target band should be clearly visible and not too close or adjacent to the nearby bands. In the gel extraction process, cutting gels of similar size may cut the gel with another close band, which may have a consequence on the accuracy of reading the sequencing results. 


In order to provide customers with the most accurate and precise sequencing results, the laboratory can accept unpurified PCR product samples with a total volume of DNA less than 50 ul, but the sample quality must be within the acceptable range. In case of single-band samples, the acceptable band must be within lane 1-2, which means the sample quality is in suitable condition with a clear target band. And the other quality of samples with multiple bands must be in the range of lane 7-8, sample; quality is in good condition with clear target band and the target band is not too adjacent to close band, which can be used for purification in the next step. 


If the customer's sample quality characteristic is in lanes 3-6, that is DNA amount is too low to be detected and/or the sample quality is in lane 9, it means that the DNA is too close to another band, which is difficult to purify and may affect Sequencing result. Therefore, the laboratory does not recommend sending unpurified PCR product samples with previously characteristics for purification.


** In case the customer would like to cut the gel by themselves. Customers can cut the gel and specify the weight of the gel to the laboratory. The laboratory will perform purification in the next step.



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