DNA Modifying Enzymes & Cloning Technologies

Magigen T7 Endonclease I / T7E1 assay /T7EI assay

● Product Name: Magigen T7 Endonclease I / T7E1 assay /T7EI assay

● Catalog#: C007S

● Size: 250 U

● T7EI Price: 85 USD

● Contents

Composition                                        Vol

10×Reaction Buffer 5                        0.5   ml×1

T7 Endonuclease I                            25 µl（10U/µl）

● Advantages and Features

Magigen T7 Endonuclease I (T7E1 assay, or T7EI assay) recognizes and cleaves non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, heteroduplex DNA and more slowly, nicked double stranded DNA. The cleavage site is at the first, second or third phosphodiester bond that is 5´ to the mismatch. Magigen T7 Endonuclease can be used to detect gene mutation, SNP, TALEN or CRISPR / cas9 mutants, identify mismatched DNA, and decompose   four-way cross DNA or branched DNA, detect or cleave heteroduplex and nicked DNA; It is used to randomly cleave linear DNA for shot gun cloning, etc.

T7 Endonuclease I is a substrate structure-selective enzyme that exhibits different cleavage activities for different DNA substrates. Therefore, when cutting a specific substrate, it is necessary to explore and control the amount of enzyme and reaction time.

● Unit Definition

One unit is defined as the amount of enzyme required to convert > 90% of 1 μg of supercoiled cruciform pUC(AT) to > 90% linear form in a total reaction volume of 50 μl in 1 hour at 37°C.

● Storage Conditions and Expiration

Sealed and stored at -20 ℃,   12 months.

● How to use T7E1?

1. Amplify DNA fragments with mismatch sites by PCR.

1.1 PCR reactions are performed using the following two templates:

gDNA from targeted cells (such as CRISPR/Cas9 or TALEN transfected cells);

gDNA from negative control cells (e.g., non-specific DNA transfected cells);

Use 100 ng of transfected cell genomic DNA as template. It is recommended that the length of amplified fragment is about 1KB, and the mutation site should not be in the middle of the fragment when designing primers.

1.2   Use commercial kits or other methods to purify PCR products while accurately quantifying them.^*1

● T7 endonuclease I digestion reaction

2.1 configure the reaction system according to the table below:

Composition                                  Vol

PCR Products                               200 ng

10×reaction buffer                      5 2 µL

Nuclease-free Water                   Up to 19 µL

2.2 the PCR products were annealed according to the following conditions:

Initial denaturation        95℃          5min

Annealing                      95-85°C     -2 °c/s

Annealing                      85-25°C     -0.1°C/s

                                      4℃             ∞

2.3 Add 1 µl T7E1   to the annealing product and incubate at 37 ℃ for 15min.

2.4 Carry out agarose gel electrophoresis to detect the digested products.^*2

*1. if it is not necessary to calculate the mutation rate and only carry out mutation detection, the PCR product can be directly annealed. If it is not purified or extracted, it is recommended that the volume of PCR product directly added to the enzyme digestion reaction should not exceed 5 µL.

*2. If the product needs to be stored for a long time after the reaction, 20mm EDTA of final concentration should be added to terminate the reaction. After 30min of storage at room temperature, non-specific cleavage phenomenon will appear.

●Note

1. Magigen T7E1 is a substrate structure selective enzyme that exhibits different cleavage activities for different DNA substrates. Therefore, when cleaving specific substrates, we must explore and control the amount of enzyme and reaction time.

2. When the reaction temperature is more than 42 ℃, the reaction time is more than 30min, and the amount of enzyme is too large, non-specific nuclease activity will appear.

3. The enzyme activity decreased significantly when the reaction temperature exceeded 55 ℃.

*All Pictures shown are for illustration purpose only and shall not form part of any offer, contract.

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