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.
| Catalog # | Pack Size |
|---|---|
|
DT0502.100 |
100 reactions (200 µl) |
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:
1. Pipettes
2. Disposable filter tips
3. 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.
Quality Control Data
Please see the certificate of analysis for each lot.
| Document | Download Link |
|---|---|
|
DTI ExoSAP Reagent – Protocol |
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DTI ExoSAP Reagent – Certificate of Analysis |
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DTI ExoSAP Reagent – Certificate of Analysis |
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DTI ExoSAP Reagent – Promotional Document |
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.