Genomic DNA was isolated from 5 × 106 to 10 × 106 mast cells using the DNeasy Blood & Tissue Kit (Qiagen). T7 endonuclease I assay to screen for mutations at selected targeted genes was carried out as previously described47 (link). Briefly, PCR primers (Supplementary Table 2 ) were designed to amplify a DNA fragment of ~1000 bp around the area targeted by the gRNAs using the high fidelity KOD Hot Start DNA polymerase (Novagen). Then, 15 µl of the PCR reaction were denatured and reannealed. Finally, 100 U of T7 endonuclease I enzyme (New England Biolabs) were added to each reaction, incubated at 37 °C for 15 min and run on an agarose gel to assess the extent of digestion of the parental band.
T7 endonuclease 1 enzyme
Manufactured by New England Biolabs
Sourced in United States
T7 endonuclease I enzyme is a DNA structure-specific endonuclease that recognizes and cleaves Holliday junctions and other DNA structures. It is commonly used in genetic research and analysis applications.
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Lab products found in correlation
4 protocols using t7 endonuclease 1 enzyme
1
T7 Endonuclease I Assay for CRISPR Mutations
2
Quantifying Genome Editing Efficiency
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After 48 h of transfection and hypoxia treatment, cells were harvested, and genomic DNA was extracted using the PureHelix™ Genomic DNA Prep kit. The target locus was amplified using an AccuPower® PCR PreMix kit (Bioneer). The obtained product was denatured by incubating at 95 °C for 5 min and reannealed by decreasing the temperature at a rate of 1 °C/1 min until it reached room temperature. Five microlitres of the sample was mixed with 1 µl of 10× buffer and 0.5 µl of T7 endonuclease I enzyme (NEB) and incubated for 1 h at 37 °C. The product was resolved on a 10% TBE gel, and staining was performed using SYBR™ Safe DNA Gel Stain (Invitrogen). Then, the staining was visualized with a Gel doc and quantified using ImageJ software.
3
T7 Endonuclease I Assay for Detecting DNA Mutations
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For the T7EI assay, the 1200-bp fragment was purified by PCR purification kit (Qiagen, Germantown, MD, USA) and submitted to the assay with T7 Endonuclease I enzyme (New England Biolabs, Whitby, ON, Canada), which recognizes and cleaves non-perfectly matched DNA. As a positive control, a 1:1 mixture of wild-type and mutant DNA fragment was used.
Briefly, 200 ng of founder’s PCR fragment or the mixture of mutant and wild-type DNA were denatured at 95°C for 10 minutes, followed by annealing during cooling at –2°C per second until 85°C, and slow cooling at –0.1°C per second until 25°C. After the formation of DNA heteroduplexes, they were incubated with 5 U of T7EI enzyme at 37°C for 30 minutes. Then, the reaction was resolved by 1% agarose gel electrophoresis. The undigested fragment must contain 1200 bp and after cleavage, bands of approximately 500 bp and 700 bp are released.
Briefly, 200 ng of founder’s PCR fragment or the mixture of mutant and wild-type DNA were denatured at 95°C for 10 minutes, followed by annealing during cooling at –2°C per second until 85°C, and slow cooling at –0.1°C per second until 25°C. After the formation of DNA heteroduplexes, they were incubated with 5 U of T7EI enzyme at 37°C for 30 minutes. Then, the reaction was resolved by 1% agarose gel electrophoresis. The undigested fragment must contain 1200 bp and after cleavage, bands of approximately 500 bp and 700 bp are released.
4
CRISPR Gene Knockout Efficiency Analysis
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Neuro-2a cells were transfected with either the control or CRISPR NSG2 KO gRNA plasmids and allowed to express the constructs for 48–60 h. The cells were then harvested and a purified GFP+ cells population was obtained by flow cytometry. The genomic DNA from the GFP+ cell population was extracted using a commercially available kit (Zymo Research); 100 μg of genomic DNA was used as a template for PCR amplification of a fragment surrounding the putative gRNA cleavage site using the following primer pair: Fwd 5’-TCCCCGGACAATGGGAATCATG-3’ and Rev 5’-GTGGCTGGAAGAATGAAAGGAT-3’. Amplicons were then subjected to a single cycle of denaturation and renaturation to generate heteroduplex molecules containing mismatches which could be recognized and cleaved using the T7 Endonuclease I enzyme (New England Biolabs). The products of the reaction were resolved on a 2% agarose gel containing 1× gel red stain (Biotium) and imaged on a gel documentation system (Bio-Rad). The relative band intensities of the cut fragments to the uncut fragment were used to calculate the gRNA-mediated cleavage efficiency.
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