||Price per sample
||2.5 - 25 kb
||25 - 125 kb
||125 - 300 kb
||600 bp - 25 kb
||25 - 125 kb
Incorrect concentration is the leading cause of failures. Kindly submit your standard, substantial, or oversized plasmid samples adhering to the specified concentration and minimum volume provided in the table above. Quantify the samples using a Qubit or an equivalent fluorometric method, such as a plate reader. We do not recommend using Nanodrop for quantification because it is not reliable enough for the sequencing equipment.
Meticulous and precise quantification, followed by proper normalization, is paramount for successful sequencing. Over or under concentrating samples will most likely have an adverse impact on the results.
For optimal outcomes, strive for the presence of pristine, circular double-stranded plasmids. Plasmids that exhibit degradation or fragmentation pose a higher risk of sequencing failure, as they may fail to yield a consensus due to a lack of complete sequencing reads.
To verify size, conduct assessments on full-length plasmids (not digested or amplified) through gel electrophoresis. For linearized plasmids, employ a linear ladder, while intact circular plasmids benefit from a supercoiled ladder. It's essential to note that Sanger sequencing and PCR amplification are insufficient for size verification since these methods rely on primers to detect specific small regions.
The workflows for significant plasmids (25 - 125 kb) and extensive plasmids (125 - 300 kb) exhibit greater resilience to degradation, given the inherent difficulty in extracting plasmids of these larger sizes without some degree of degradation. Nonetheless, to ensure optimal results, the objective remains intact circular DNA even within these more forgiving workflows.
The linear/amplicon workflow exhibits greater resilience to degradation compared to the plasmid workflow, owing to its incorporation of minimal fragmentation during the library preparation process. Nevertheless, to achieve optimal outcomes, the pursuit of intact linear DNA molecules remains a paramount consideration.
The cleaner the product, the better the results. Leftover material in the sample can act as an inhibitor during sequencing. We suggest opting for samples exhibiting a 260/280 ratio exceeding 1.8 and a 260/230 ratio falling within the range of 2.0-2.2. Purity assessments can be conducted using Nanodrop or spectrophotometric methods, although these methods cannot be used to accurately measure the concentration.
For optimal outcomes, it is imperative that samples do not include any of the following components:
- Denaturants, such as guanidinium salts, phenol, etc., or detergents like SDS, Triton-X100, etc.
- Leftover contaminants from the organism.
- RNA (we recommend RNase treatment during extraction for its removal)
- Any insoluble material that would cause colors or cloudiness.
Furthermore, samples should contain copies of a single clonal molecule. You can send mixtures but it may yield inconsistent results. Submit mixtures at your own risk.
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