DTI ExoSAP Reagent
DTI ExoSAP Reagent
DTI brand product DTI ExoSAP Reagent is designed to perform enzymatic cleanup of amplified PCR product enabling downstream applications, primarily Sanger sequencing. The product is a combination of enzymes Exonuclease and Shrimp alkaline phosphatase in optimal ratio currently available in two pack sizes of 200 µl for 100 reactions and 1 ml (200 µl x 5) for 500 reactions.
SKU:DT0502.100
DTI ExoSAP Reagent
| Brand | Catalogue Number | Pack Size |
|---|---|---|
| DTI ExoSAP Reagent | DT0502.100 | 100 reactions (200 µl) |
| DTI ExoSAP Reagent | DT0502.500 | 500 reactions (200 µl x 5) |
- Principle:
To achieve high-quality sequencing results, it is crucial that the PCR product is free from unused/excess primers and dNTPs in the PCR product that could interfere with the sequencing reactions and lead to high background. The enzyme Exonuclease in the DTI ExoSAP Reagent degrades single-stranded DNA (including oligonucleotide primers), while enzyme Shrimp alkaline phosphatase degrades the Nucleotides to nucleosides and Inorganic phosphate. Both enzymes are subsequently removed by heat-inactivation and DNA is ready for sequencing.
- Storage: -20°C
- Components:
Components Cat# DT0502.100 (volume) Cat# DT0502.500 (volume) DTI ExoSAP Reagent 100 reactions (200 µl) 500 reactions (200 µl x 5) - Materials required but not provided:
- Pipettes
- Disposable filter tips
- Incubator
- Protocol:
- Remove DTI ExoSAP Reagent from -20°C freezer and keep on ice throughout this procedure.
- Add 2μl of DTI ExoSAP Reagent directly to 5 μl PCR product and mix well.
- Incubate for 10 mins at 37°C to degrade remaining primers and nucleotides.
- Incubate for 5 min at 85°C to inactivate the enzymes.
- PCR products are ready for downstream applications without any need for further processing.
- Quality Control Data: Please see the certificate of analysis (CoA) for each lot.
DTI ExoSAP Reagent - Protocol and Downloads
| Document | Download Link |
|---|---|
| DTI ExoSAP Reagent - Protocol | Download User Manual |
| DTI ExoSAP Reagent - Certificate of Analysis | Download CoA - DT0502.100 |
| DTI ExoSAP Reagent - Certificate of Analysis | Download CoA - DT0502.500 |
| DTI ExoSAP Reagent - Promotional Document | Download Flyer |
Comparison of Sanger Sequencing Quality: DTI ExoSAP Clean-up vs. Untreated PCR Product Sequencing
PCR product was subjected to enzymatic clean-up by the DTI ExoSAP reagent and further subjected to Sanger sequencing. This was compared against a PCR product that was directly sequenced without clean-up. The quality of base calling is better in the PCR product that was subjected to clean-up by DTI ExoSAP reagent as compared to the product that was directly sequenced. The electropherogram images of both samples are as follows:
Figure 1: Electropherogram showing quality of sequencing after DTI ExoSAP clean-up vs untreated PCR product.
High-quality sequencing data after treatment with DTI ExoSAP reagent is obtained. Bars above sequence represent the quality value of the assigned base. An assigned base with QV = 20 has an error probability of 0.01. High-quality peaks generally have a QV of 20 or higher. Blue bars represent QV >20, Yellow bars represent QV between 15-20, and Red bars represent QV <15.