Exo-sap Cleanup
Exo-sap Cleanup - Related Videos
09:27
The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision
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Cited by 9 •
2016
Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.
11:23
Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes
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Cited by 13 •
2018
Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.
10:14
Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects
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Cited by 69 •
2018
This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.
08:40
High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)
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Cited by 2 •
2020
Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.
07:27
Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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Cited by 11 •
2010
The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision.
FAQs
The two enzymes target different PCR carryover components. Exonuclease I degrades unincorporated single-stranded primers, while shrimp alkaline phosphatase hydrolyzes residual deoxynucleotide triphosphates, or dNTPs. Combining these activities produces a more complete cleanup than addressing only one contaminant, which helps prepare amplified DNA for reliable downstream sequencing and mutation analysis.
Unremoved primers and dNTPs can interfere with subsequent sequencing reactions, potentially reducing the accuracy of results obtained from an amplified DNA product. ExoSAP cleanup removes these residual components before downstream analysis, helping researchers interpret sequence data, mutation calls, or genotyping results with less interference from materials carried over from PCR.
Controlled incubation gives exonuclease I and shrimp alkaline phosphatase time to act on their respective PCR residues. A subsequent enzyme-inactivation step stops their activity before the cleaned amplification product enters downstream analysis. This sequence preserves the intended cleanup while preparing the DNA for uses such as Sanger sequencing or mutation validation.
The workflow begins with an amplified DNA product containing residual primers and dNTPs. The cleanup enzymes are allowed to act during a controlled incubation, after which the enzymes are inactivated. The treated product can then proceed to downstream analysis without the extensive purification steps otherwise used to remove these PCR-associated contaminants.
ExoSAP cleanup is useful when a PCR product must be prepared efficiently for downstream analysis and the main contaminants are unincorporated primers and dNTPs. Because enzymatic treatment addresses these residual components without extensive purification, it can streamline workflows involving Sanger sequencing, mutation analysis, genotyping, and validation of amplification products.
In cancer research, the cleaned PCR product can support characterization of genetic alterations in tumor samples and cancer-related models. By reducing contaminants that may interfere with sequencing reactions, the method contributes to accurate Sanger sequencing, mutation analysis, genotyping, and validation of amplified DNA, helping researchers evaluate cancer-associated genetic changes.