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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.

Specifications

Documents

Applications

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:

1. Remove DTI ExoSAP Reagent from -20°C freezer and keep on ice throughout this procedure.

2. Add 2μl of DTI ExoSAP Reagent directly to 5 μl PCR product and mix well.

3. Incubate for 10 mins at 37°C to degrade remaining primers and nucleotides.

4. Incubate for 5 min at 85°C to inactivate the enzymes.

5. 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

DTI ExoSAP Reagent - Certificate of Analysis

DTI ExoSAP Reagent - Promotional Document

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 which was subjected to clean-up by DTI ExoSAP reagent as compared to the product that was directly sequenced. The electropherogram images of both samples is as follows:

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

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