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Questions tagged [restriction-enzymes]

Endonucleases are enyzmes (usually derived from bacterial sources) which cleave DNA at defined "restriction" sites.

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Double Digestion with Restriction Enzymes Using Different Buffers

I am currently working on preparing a 9 kb sequence of DNA for restriction digestion into the pBAD30 expression vector. There are very few restriction enzymes that do not have a restriction site ...
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What restriction enzyme was used in cutting this DNA piece? [closed]

So, i was given this cut piece of dna and i was required to choose the restriction enzyme used to cut it. I had to pick either XhoI(CTCGAG) or TaqI(TCGA), where the sequence between parentheses is ...
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How do I reverse my insert within a plasmid?

I have a plasmid with: ...T7pro. -- RBS -- XBa1 cut site -- ProDpro. -- RBS -- AmilCP -- T -- Spe1 cut site -- T... How do I design primers to reverse the portion in bold, such that the T7 and ProD ...
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Cutting of DNA strand [closed]

Would EcoRI cut the following sequence?? 😕 5'CTCGAGTTCGAG3' 3'GAGCTCAAGCTC5' What is the logic of cutting? Please explain how restriction enzymes work.
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Where is cleavage site located? [closed]

I feel a bit confused about cleaving! Is cleavage site always located on the substrate or on the enzyme?
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Cutting dna using restriction endonuclease

Genomic dna is digested with alu 1 which is a four base pair cutter. What is the frequency with wich it will cut the dna assuming a normal distribution of bases. (this is not a homework)
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Why does DNA need to be pure for restriction enzymes to act on it?

My textbook states: “In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules. Therefore DNA isolated from other chemicals of the protoplasm like ...
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Cloning problem. Digestion test at the end of the cloning is always negative

I have a problem with my cloning, I'm trying to clone a 483 pb in a pSecTag vector with restriction sites HindIII and BamHI. Here are the procedures I'm doing: • I introduce the restriction sites ...
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What does the end of DNA look like?

EcoR I ........ 5'-GAATCC-3' BamH I ....... 5'-GGATCC-3' Hind III........ 5'-AAGCTT-3' The enzyme BamHI recognizes a palindromic sequence and leaves one strand longer than the other. A ...
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Restriction enzyme type II [closed]

Why can't a type II restriction enzyme cut a nucleic acid with the following base composition: A: 28% C: 15% G: 35% T: 22%
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Why don't restriction endonucleases digest transformed plasmids?

In the textbook that I'm using, it explains that bacteria does not digest its own chromosomal DNA because the sites that would be cut by its own endonuclease are methylated. Is there a similar ...
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Why is EcoRI supplied with a unique buffer when it is allegedly 100% active in universal buffers?

Thermo packages a unique buffer with EcoRI: 50 mM Tris-HCl (pH 7.5) 10 mM MgCl2 100 mM NaCl 0.02% Triton X-100 0.1 mg/mL BSA However, they also list their EcoRI enzyme as 100% active ...
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Why doesn’t p53 cause the repair of cellular DNA that has been altered experimentally?

When scientists mutate bacteria or even embryos of lambs and other animals, why doesn't the p53 reverse whatever mutation the scientists cause? I know that the p53 stops the DNA from mutating and ...
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What are the possible reasons to get extra unrecognized band in agarose gel electrophoresis?

I ran 0.7% agarose gel electrophoresis of my plasmid DNA sample consisting of GFP vector, using NheI and HindIII restriction enzyme. I used two types of plasmids, i.e., isolated by miniprep-alkali ...
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Will two Type IIS restriction sites cut if they are directly back-to-back?

I want to put two BbsI (or BsaI) restriction sites right next to each other, facing out, like this: ...
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1answer
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Plasmid specific DNA degradation

I'm interested if there is a way to degrade the plasmid DNA inside an E.Coli cell specifically so that the method does no harm to the chromosomal DNA. First I was thinking about restriction ...
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Standard graph - Determining sizes of restriction fragments

I have marked out Lane M and gotten a roughly straight line, it then says that I am supposed to estimate the length of lane H and R. I am not sure what they are asking. I am still supposed to measure ...
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Is a Linker sequence mandatory in primers for PCR Cloning?

I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no ...
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Is blunt end repair and A-tailing necessary when adaptors can be custom made?

I'm planning on creating a RADseq library, which naturally involves digesting genomic DNA following P1 adaptor ligation. I've read a few protocols that require any over-hangs resulting from ...
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Has a restriction enzyme ever been tagged?

As part of an experiment that I am preparing, it would seem necessary for me to tag a restriction enzyme (HaeIII to be exact) with GFP. I began researching its domains to determine whether to tag the ...
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Restriction enzymes, how are the recognition sequences determined?

How were the recognition sequences (e.g. GAATTC of EcoRI, GGATCC of BamHI) characterised? Text books only list the recognition ...
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How to cut out a specific named gene from plant DNA

Suppose one has extracted RNA from a plant, converted it to the corresponding cDNA & amplified it but now wants to cut out a particular, already-sequenced gene out from it, how does one proceed? ...
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RAD sequencing: choosing the appropriate enzyme?

I’m studying Darwin’s finches genome and I say in some articles that the researchers used restriction enzymes to cut the DNA in their double digest RAD protocol. They are using EcoRI and MseI (GAATTC ...
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1answer
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Enzymes and plasmids

A high school class has analyzed plasmids by the help of restriction enzymes and electrophoresis. The class got delivered two different plasmids, pBR 322 and pC 508. These two plasmids were going to ...
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1answer
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What happen if we inject restriction enzyme into the blood

Just a curious question, if we extract restriction enzyme and inject it to our body, what happen? Does antibody recognize and block it? Can restriction enzyme pass over cell membrane and destroy the ...
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Freezing after restriction digest

Brief question: I have performed a double digest with heat inactivated restriction enzymes with no star activity. I usually purify the DNA (PCR inserts and linearised vector) using a kit or gel ...
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Batch several sequences for absent restriction sites

I have a collection of about 120 7kB sequences I would like to check for ether a list of specific restriction sites, or what restriction sites might be absent in all of them. Is there a app or ...
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Restriction sites

I would like to know: how many restriction sites does a restriction enzyme use on a DNA molecule? In other words: If a sequence on a plasmid contains the following bases: ATTGCAGTCTG and I want to ...
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Cleavage of RNA by restriction enzymes?

Six restriction enzymes discussed in Sequence-specific cleavage of RNA by Type II restriction enzymes (Murray et al.) have the ability to detect and cut RNA strands with that enzyme's recognition ...
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1answer
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Do restriction enzymes on read 3' to 5'?

Every chart of palindromic restriction enzymes I've seen lists their restriction sites from 5' to 3', something like this: EcoR1 cuts GAATTC between the G and A: 5' NNNGAATTCNNN 3' --> ...
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1answer
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Does mung bean nuclease cleave a phosphate group when it's chewing off 5' or 3' ssDNA ends?

I'm looking to create blunt ends from sticky ends with mung bean nuclease for subsequent ligation. Does anyone know full mechanism by which mung bean nuclease will do this? In particular after the ...
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Are restriction enzymes active at −20 °C?

I have digested my DNA with NotI enzyme and put it in the −20 °C freezer without heat inactivating it. Can restriction enzymes work at −20 °C? Should I expect STAR activity?
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What makes DNA sticky-ends sticky?

When restriction enzymes jaggedly cut double stranded DNA it results in so called sticky ends. What is the substance that makes the DNA sticky?
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Molecular Cloning- Blunt end restriction endonucleases

I work in a microbiology lab where we do a lot of cloning. I have always used restriction endonucleases to cleave the DNA to have sticky ends and not blunt ends. I currently am working on a project ...
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How many Type II restrictions enzymes are currently available (commercially) [closed]

How many Type II restrictions enzymes are currently available (commercially) for purchase as of the date of this posting (September 1, 2014)
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How do Type I restriction enzymes work?

According to NCBI, type I REases have a specific target sequence but randomly make cuts at sites far from the target sequence. How does the restriction enzyme travel from the target sequence to the ...
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1answer
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What does 'H' in BamHI stand for?

It's not explained on the wikipedia page. So if, apparently, 'H' is not relevant, why is it part of the name? And if it is relevant, why is it not explained?
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How was Restriction Site of EcoRI sequenced?

The sequence of restriction site of EcoRI - GAATTC was identified in the early 1970s, before Sanger Sequencing was invented.(1977) How was the restriction site of EcoRI sequenced ?
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Restriction endonucleases are found in?

Quoting from : Scientific American July 1975 The Manipulation of genes by Stanley Cohen : Restriction endonucleases (and modification methylases) are widespread in microorganisms; genes for ...
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Removing a restriction site and introducing other at its place

What would you do if you want to remove an EcoRI restriction site and introduce BamHI restriction site at apprx. the same location ? One of the answers in my textbook was : To construct a DNA ...
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DNA fingerprinting

I would like to make my own DNA fingerprint - just for fun to have my "autoportrait":). I was looking around a bit and all the commercial kits you can have are very expensive. Can you suggest me a ...
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Gel electrophoresis question

Leena is a molecular biology student. She purifies two fragments of DNA, 800 and 300 base pairs long. These were obtained from a plasmid after digesting it with HindIII. Each of these fragments has a ...
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How effective are restriction enzymes in protecting bacteria?

Bacteria use restriction enzymes to cut DNA of bacteriophages. Virus mutates really fast. Won't a point mutation in restriction site render the restriction enzymes of the bacteria useless ? So how ...
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Why do Type II Restriction Endonucleases cleave at palindromic sequences?

Type II Restriction enzymes usually cut only at palindromic sequences. Is there any specific reason for that? Is there any advantage for bacteria if they cleave phage DNA at this type of sequence?
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What is the advantage of restriction enzymes cutting only at specific sites?

Bacteriophages have sequences which often do not have specific sites for restriction enzymes of bacteria to cut at and so can attack the bacteria. Wouldn't it be better if bacteria had something "...
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What can cause incompatible sticky ends to be ligated?

Actual question I have reason to believe (details see below) that in a ligation I carried out, an EcoRI sticky end (EcoRI: G'AATT_C) and an XmaI sticky end (XmaI: C'CCGG_G) were somehow ligated ...
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Can viruses protect themselves against restriction enzymes?

Restriction enzymes cut the DNA of bacteriophages. Have bacteriophages evolved any mechanism to protect themselves from it ?
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What are common causes of unexpected ligation products?

I digested two plasmids, one with EcoRI and AgeI and the other with EcoRI and XmaI. Digests looked as expected, so I purified the respective fragments and set up the T4 DNA ligation (AgeI and XmaI ...
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2answers
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How prevalent is Taq polymerase in adding 3' A overhangs to the PCR product?

I am conducting a mutagenesis on a gene in vivo of which I need to ligate into an expression vector. The primers I have designed overlap restriction sites of which I plan to use to ligate into the ...
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Restriction Mapping of Plasmid Assignment

pUWL22 is a circular, double stranded, 10.5 kb plasmid. It contains a gene encoding an enzyme that confers ampicillin resistance in the host bacterium. Cloning into the kpn I and Sst I sites ...