≥95%, This product is free from RNase, phosphatase, and other DNA exonucleases and endonucleases.
Stability And Storage
Store at -20℃, valid for at least 1 year.
Storage Temp
Store at -20°C
Shipped In
Ice chest + Ice pads
Product Description
Aladdin's T7 Endonuclease I is a special DNA endonuclease I that recognizes and cleaves non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, and heteroduplex DNA. It can also cleave nicked double-stranded DNA more slowly. The cleavage site is at the first, second, or third phosphodiester bond that is 5´ to the mismatch. This product is generally used for detecting genome mutations introduced by CRISPR/Cas9 or other gene editing technologies.The cleavage of mismatched base sites by T7E1 is shown in Figure 1. It should be noted that T7E1 can recognize mutations greater than or equal to 2bp, but not 1bp mutations. Figure 1
Application：
T7E1 is commonly used to detect mutations introduced by CRISPR/Cas9, TALEN, or other gene editing technologies, and to detect SNP sites. It can also be used to resolve four-way junction or branched DNA, detect or cleave heteroduplex and nicked DNA, and randomly cleave linear DNA for shot-gun cloning and sequencing.Please see Figure 2 for the cleavage of heteroduplex dsDNA by ’s T7E1 enzyme : Figure 2: Agarose gel electrophoresis of dsDNA with or without treatments of T7E1 . A 800bp DNA fragment (wild type and mutant) is amplified using a DNA polymerase with high fidelity. Equal amounts of wild-type DNA and DNA mutant (100ng each) were mixed together to generate heteroduplex by denaturation, annealing, and renaturation (95℃ 5min, 95℃-8℃C -2℃/second, 85℃-25℃ -0.1℃/second). Wild type DNA, DNA mutant, and heteroduplex (200ng each) were digested by 10U T7E1 at 95℃for 30 minutes, and then examined by 1% agarose gel electrophoresis. The results demonstrates that only heteroduplex DNA is cleaved by the T7E1 into 600bp and 200bp fragments, and T7E1 has comparable performance to Competitor product.'s T7E1 is compatible with a variety of PCR amplification systems (Figure 3). The PCR product can be added directly into T7E1 reaction without any prior preparations. Figure 3: Agarose gel electrophoresis of Control Template cleaved by T7E1 in the presence of different PCR buffers. Same amount of Control Template was added into different 1X PCR buffers. After mixing, take 5µl of each and incubate with 10U T7E1 at 37°C for 30min in a total reaction volume of 20µl, respectively. The digestion results were examined by 1% agarose gel electrophoresis. 1. No T7E1；2. T7E1 with no PCR buffer; 3. T7E1 with D7205-2 Buffer；4. T7E1 with D7218-2 Buffer；5. T7E1 with D7216-2 Buffer；6. T7E1 with D7220-2 Buffer；7. T7E1 with D7211-2 Buffer；8. T7E1 with D7213-2 Buffer；9. T7E1 with D7215-2 Buffer；10. T7E1 with D7241S-2 Buffer；11. T7E1 with D7243S-2 PCR Buffer；12. T7E1 with D7248S-2 Buffer. The results demonstrates that T7E1 is compatible with all these tested PCR buffers. Therefore, up to 5µl of PCR product with single band can be added directly into a 20µl of T7E1 reaction without any prior preparations.
Source：
Recombinant T7 Endonuclease I expressed in E. coli.
Inactivation or inhibition：
Incubation at 85ºC for 15 minutes, or addition of EDTA to a final concentration of 20mM.
Precautions：
T7E1 is a structure-dependent enzyme that performs selective digestion and displays different activity on different DNA substrates. Therefore, it is necessary to optimize the amount of T7E1 and incubation time for a specific DNA substrate.Non-specific digestion by T7E1 increases when the temperature is above 42ºC. T7E1 enzyme activity decreases when the temperature is over 55℃.This kit is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
1. Thaw all reagents at room temperature and keep on ice.2. Assemble the reaction (20μl) in the following order:ComponentPositive ControlNegative ControlControl templatePCR product(~200ng)5 μl5 μl2 μl10 x T7 Endonuclease I Reaction Buffer2 μl2 μl2 μlNuclease-free Water12 μl12 μl15 μlNote: We recommend using 5μl of PCR product. Addition of more PCR products may interfere with the subsequent enzyme digestion due to the presence of PCR buffer components.3. Annealing.StepTemperatureTime195℃5min295°C–85℃–2℃/sec385°C–25℃–0.1℃/sec44℃Hold4. Add 1µl of T7E1 into the reaction, mix gently, and pulse-spin in a microfuge. 5. Incubate at 37℃ for 15-30 minutes. Generally, 15 minutes is sufficient.6. Stop the reaction by incubation at 85ºC for 15 minutes, or by adding 1.5µl of 0.25M EDTA.7. Proceed with fragment analysis by agarose gel electrophoresis.
