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T7 Endonuclease I

No Product Name Cat.# Conc Size Price(₩) Add
1 T7 Endonuclease I M048S 10 unit/μl 250 units ₩ 80,000 추가
2 T7 Endonuclease I M048L 10 unit/μl 1,250 units ₩ 320,000 추가
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Product description

T7 Endonuclease I (T7 Endo I), the resolvase from T7 phage of Escherichia coli, belongs to the endonuclease family. This product is an engineered form of T7 Endo I. It recognizes and cleaves mismatched DNA, cruciform DNA, heteroduplex DNA, four-way junctions, as well as nicked double-stranded DNA. The site of cleavage is at the first, second or third phosphodiester bond that is 5´ to the mismatch.

 

Characteristics

- The resolvase from T7 phage of bacterium Escherichia coli

 

Applications

- Mutation detection assay

- Detection and cleavage of heteroduplex or nicked DNA

- Linear DNA cleavage for shotgun cloning

 

Quality control

- Endonuclease-free

- Exonuclease-free


Heat inactivation

No


Supplied with

- 10X EzBuffer II

- Sterile water

 



Figure 1. Assaying the formation of digestion product by T7 Endonuclease I

Digestion mixtures containing heterozygous mutant and T7 Endonuclease I were incubated at 37 for 15 min. Reaction products were analyzed on 1.5% agarose gel and stained with EtBr.

● Manual download

- Enzynomics_M048_T7 Endonuclease I_manual

 

● Measurement of genome targeting efficiency using T7 Endonuclease I

T7 Endonuclease I recognizes and cleaves DNA that does not match perfectly. This protocol describes how to measure genomic targeting efficiency by digesting PCR products with T7 Endonuclease I.

 

1. PCR

1-1. PCR mixturea

Component

50 µl reaction

Final Concentration

2X TOPsimple™ DyeMIX–Forte

25 µl

1X

10 µM Forward Primer

2.5 µl

0.5 µM

10 µM Reverse Primer

2.5 µl

0.5 µM

Template DNA

variable

100 ng total

Sterile water

To 50 µl

 

aAssmeble the reaction mixture on ice

 

1-2. PCR cycles

Step

Temperature

Time

Cycle

Initial denaturation

95

2 min

1

Denaturation

95

30 sec

25~35

Annealing

55~65

30~60 sec

Elongation

72

1 min/kb

Final elongation

72

5 min

1

 

2. T7 Endonuclease I digestion

2-1. Substrate prepares

Substrate DNA (PCR product)

200 ng

10X EzBuffer II

2 μl

Sterile water

Up to 19 μl

 

2-2. Hybridization Conditions

Initial denaturation

95

5 min

Annealing

95~85°C

-2°C/sec

85~25°C

-0.1°C/sec

Hold

4

Hold

 

2-3. T7 Endonuclease reaction

Annealed PCR product

19 μl

T7 Endonuclease I

1 μl

→ Incubate 37, 15 min

→ Stop the reaction by adding 1.5 µl of 0.25 M EDTA.

 

3. Analysis

- Analyze fragmented PCR products and determine the percentage of nuclease-specific cleavage

products (cleavage cleavage)

- Calculate the estimated genetic variation using the following formula:

% gene modification = 100 x (1 – (1- fraction cleaved)1/2)

 


● Material Safety Data Sheet

Enzynomics_MSDS_M048_T7 Endonuclease I_E

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