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CRISPR Mutation Detection Kit

$145.00$349.00

The CRISPR Mutation Detection Kit is useful for quantitatively estimating the nuclease-induced mutation frequency of gene edited cells.


    25 Reactions - $145.00 (Cat.# 3173 )

    100 Reactions - $349.00 (Cat.# 3176 )

Description

Double-stranded breaks (DSBs) generated by CRISPR/TALEN/ZFN at desired target sites can be PCR-amplified, and the PCR products can be denatured and re-annealed to form mismatched DNA. If the mismatched DNA length position is more than 1 bp, T7 endonuclease I can recognize and cleave it. It is useful for quantitatively estimating the nuclease-induced mutation frequency of gene edited cells.

Product Includes

  • i7™ high fidelity 2x PCR master mix
  • T7 Endonuclease I
  • 10x T7 Endonuclease I reaction buffer

Storage Temperature: –20 °C

Please view the “Protocol” tab to the left for usage and troubleshooting information.

1013-13 1013-36

Additional information

Reactions

25 Reactions, 100 Reactions

Protocol
This protocol describes how to determine genome targeting efficiency by digesting annealed mismatched PCR products with T7 Endonuclease I. In the first step, PCR products are produced from the genomic DNA of cells whose genomes were targeted using Cas9, TALEN, ZFN etc. In the second step, the PCR products are annealed and digested with T7 Endonuclease I. If two shorter fragments of the expected size are generated, which means that it has successfully introduced mutations at the targeted chromosomal site. Fragments are analyzed to determine the efficiency of genome targeting.

CRISPR Detection Kit Protocol

Procedure

I. Sample Preparation

Option 1: Genomic DNA extraction

Harvest cells and extract genomic DNA according to the manufacturer extraction protocol.

Option 2: Cell lysate preparation

Collect cells and add 50-100 μL Lysis buffer and lyse cells at 95º C for 5-10 min.

II. PCR Amplification

  1. Thaw kit components, mix and pulse-spin in micro-centrifuge before use.
  2. Assemble the following reaction:

CRISPR Detection Kit Reaction Set up

  1. Mix the reaction and run PCR with the following thermocycling conditions:

CRISPR Detection Kit PCR Cycling Conditions

  1. Run 3-5 μL of the PCR product on a 1-2 % agarose gel. The final PCR product should be within the range of 600 –1000bp.
  2. (Optional) If necessary, purify the DNA by using either gel extraction kit or ampure XP beads according to the manufacturer protocol

III. Hybridization

  1. Assemble reactions as follows:

CRISPR Detection Kit Hybridizaton set up

  1. Mix the reaction gently.
  2. Heat at 95º C for 5 min by using a heat block. Thermocyler can also be used for hybridization.
  3. Turn off the heat block and cool down gradually to room temperature.

IV. T7 Endonuclease I Digestion

  1. Assemble reactions as follows:

CRISPR Detection Kit Endonuclease Digestion

  1. Incubate at 37º C for 15-30 min.
  2. Stop the reaction by adding 1.0 µl of 0.5 M EDTA.
  3. Run 1-2 % agarose gel to see the cleavage efficiency. A typical gel for Endonuclease I (Endo I) cleavage is shown below:

CRISPR Detection Kit Cleavage eff

Technical Support
Intact Genomics is committed to supporting the worldwide scientific research community by supplying the highest quality reagents. Each new lot of our products is tested to ensure they meet the quality standards and specifications designated for the product.

Please follow the instructions carefully and contact us if additional assistance is needed. We appreciate your business and your feedback regarding the performance of our products in your applications.

CRISPR Detection Kit Troubleshooting

Manual -ig® CRISPR Mutation Detection Kit

MSDS -ig® CRISPR Mutation Detection Kit

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