What is the purpose of ExoSAP?

What is the purpose of ExoSAP?

ExoSAP-IT™ PCR Product Cleanup Reagent is used for enzymatic cleanup of amplified PCR product. It hydrolyzes excess primers and nucleotides in a single step. ExoSAP-IT-purified samples are ready for use in downstream applications such as DNA sequencing or single nucleotide polymorphism (SNP) analysis.

How does ExoSAP work?

ExoSAP-IT utilizes two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase, to remove these unwanted dNTPs and primers. The Exonuclease I degrades residual single-stranded primers and any extraneous single-stranded DNA produced by the PCR.

How do you clean PCR?

There are several methods for PCR cleanup such as ethanol precipitation, bead or column based purification, and Enzymatic approaches. Ethanol precipitation is most cost effective but is also the most labor intensive. Bead or column based purification can be an effective method for many applications.

What is cycle sequencing?

Cycle sequencing is the method required to amplify and fluorescently label your DNA so that it can be detected on our automated sequencers. Cycle sequencing is very similar to PCR, but with two major differences.

How does Sanger sequencing work?

Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end. The extension products are then separated by Capillary Electrophoresis or CE. The molecules are injected by an electrical current into a long glass capillary filled with a gel polymer.

What is a PCR clean-up?

Video: PCR Clean-Up Procedure The GenElute™ PCR Clean-up Kit is designed for rapid purification of single-stranded or double-stranded PCR amplification products (100 bp to 10 kb) from other components in the reaction, such as excess primers, nucleotides, DNA polymerase, oil and salts.

What is the purpose of PCR clean-up?

The purpose of this procedure is used to chew up excess primers and remove excess dNTPs from your PCR product. This proceedure is necessary to ensure clean and readable DNA sequences.

What is the principle of Sanger’s sequencing technique?

In automated Sanger sequencing, a computer reads each band of the capillary gel, in order, using fluorescence to call the identity of each terminal ddNTP. In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light emitted.

What are the steps of DNA sequencing?

What are the steps in DNA sequencing?

• Sample preparation (DNA extraction)
• PCR amplification of target sequence.
• Amplicons purification.
• Sequencing pre-prep.
• DNA Sequencing.
• Data analysis.

Is Sanger sequencing still used?

Sanger sequencing is still widely used for small-scale experiments and for “finishing” regions that can’t be easily sequenced by next-gen platforms (e.g. highly repetitive DNA), but most people see next-gen as the future of genomics.

What is the primary disadvantage of Sanger sequencing?

Limitations of Sanger Sequencing Sanger methods can only sequence short pieces of DNA–about 300 to 1000 base pairs. The quality of a Sanger sequence is often not very good in the first 15 to 40 bases because that is where the primer binds. Sequence quality degrades after 700 to 900 bases.

Why do we do PCR clean up?

Purification of DNA from a PCR reaction is typically necessary for downstream use, and facilitates the removal of enzymes, nucleotides, primers and buffer components. Finally, the DNA is eluted from the columns under low-salt conditions and is ready for demanding downstream applications. …

Is the exosap-it reagent able to digest dsDNA?

No. ExoSAP-IT reagent does not digest dsDNA, which is why it leaves PCR products and primer dimers undigested. To eliminate primer dimer formation, we recommend that you check for optimal primer design and use PCR polymerases with hot start technology or setup reactions on ice.

When to use exosap-it for PCR cleanup?

ExoSAP-IT™ PCR Product Cleanup Reagent is used for enzymatic cleanup of amplified PCR product. It hydrolyzes excess primers and nucleotides in a single step.

How does the exosap-it one step reaction work?

The ExoSAP-IT one-step solution provides a simple way to enzymatically purify a PCR reaction. The reaction setup is complete with one pipetting step, which is followed by two incubations. The first incubation digests excess primer and dephosphorylates nucleotides.

What can exosap-it be used for in downstream applications?

ExoSAP-IT-purified samples are ready for use in downstream applications such as DNA sequencing or single nucleotide polymorphism (SNP) analysis. The ExoSAP-IT one-step solution provides a simple way to enzymatically purify a PCR reaction. The reaction setup is complete with one pipetting step, which is followed by two incubations.