Questions tagged [restriction-enzymes]
Endonucleases are enyzmes (usually derived from bacterial sources) which cleave DNA at defined "restriction" sites.
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How to understand restriction mapping a quarter of a century later?
Today, biology is virtually all based on massively parallel sequencing, long-strand sequencing, and metagenomic; looking back at old restriction mapping is not straightforward (at least for me).
For ...
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Why using Fokl as an enzyme in the zinc finger nuclease method?
I started reading about CRISPR, and then about older methods of gene editing. I recently learnt about zinc finger nucleases, where Fokl (a restriction enzyme without specificity) gets specificity, ...
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Change of DNA concentration due to restriction digest?
Assume that you perform a restriction digest in a molecular biology lab: you combine genomic DNA, a restriction endonuclease (e.g., EcoRI), and the optimal buffer for that endonuclease and are about ...
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Which was the first restriction endonuclease to be isolated?
I kind of have confusion between the first restriction endonuclease to be isolated and discovered.
In class XII NCERT, it says that,
"In the year 1963, the two enzymes responsible for restricting ...
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Database for commercially available restriction enzymes?
I need a database that contains a complete list of commercial restriction enzymes: name of the enzyme & recognition site.
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Linearising plasmids for CRISPR experiment
I am currently designing a mock CRISPR knock-out experiment, and I’m wanting to insert a plasmid for selection. Using a restriction enzyme at 2750bp for cutting, would the location of the cut site ...
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Explanation for the results of Topoisomerase I treatment of ERCs - Gel Electrophoresis
This part of a 1997 paper published in The Cell (David A. Sinclair & Leonard Guarente, 1997) discusses the presence of extrachromosomal rDNA circles (ERCs) in greater numbers in old mutant yeast ...
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Why am I losing much more vector than insert DNA during RE cloning?
I've been cloning some inserts into a lentiviral vector (pBOB backbone) by RE digestion and blunting ends with T4 DNA polymerase.
After these steps, I usually load the digested product onto 0.7% ...
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DNA Design for Multi-Site Restriction Enzymes
I have to use the SacII restriction enzyme, which requires multiple recognition sites to efficiently cut DNA, for a DNA assembly. From my understanding, multiple copies of DNA holding only a single ...
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What breaks hydrogen bonds while producing sticky ends using restriction endonucleases?
I am a high school student and I am little confused about the uses of restriction endonucleases. Why do hydrogen bonds(base pairing)
break when restriction endonucleases produce sticky ends? If they ...
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Do PI-SceI and PI-PspI from NEB work in each other's buffers?
I'm considering a plasmid design strategy that would allow me to use restriction enzymes to exchange plasmid inserts among different backbones. In this case, the plasmids would first be made through ...
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How to replace intron region in a plasmid?
I'm considering working with the plasmid pRFHUE-eGFP and would like to replace the gpdA intron (which is the eGFP promoter region) with a promoter from another organism.
What would be a good strategy ...
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What if target DNA doesn’t have restriction sites
All the examples on DNA cloning I have encountered have assumed that the target gene and vector both have compatible restriction sites at just the right locations (probably for ease of explanation). ...
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Can restriction enzymes (Type II) displace single-stranded binding proteins (SSB)?
Many type II restriction enzymes have been shown to be able to cut ssDNA.
If single-stranded binding proteins are bound to single-stranded DNA (ssDNA), does this prevent restriction enzymes which cut ...
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How to calculate the occurrence of a stretch of nucleotides in a genome?
I have seen that the formula to calculate the number of times a given sequence of nucleotides occur in a target genome is derived from that to calculate the expected frequency of restriction sites:
<...
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