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Topic: Restriction digest

	Restriction Digests (Site not responding. Last check: 2007-11-04)
		Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases (sometimes just called Restriction Enzymes or RE's).
		Restriction Digests begin by mixing the DNA and the RE, but it's unfortunately not quite as simple as that.
		Once the Restriction Digest is completed, Agarose Gel Electrophoresis is performed to separate the digest fragments by size and visualize the fragments and perhaps purify them for further experiments.
	www.life.uiuc.edu /molbio/geldigest/digest.html (244 words)

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		Analyze all digests with gel electrophoresis Introduction Recombinant DNA technology or “gene splicing,” as it is frequently called, often involves insertion of selected DNA sequences from a variety of sources into plasmids which are then transformed into bacteria.
		Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by hydrolyzing the phosphodiester bonds between the nucleotides.
		Restriction Enzyme Digestion of pRAS2 DNA (note- all components must be kept on ice) 1.
	www-hhmi.princeton.edu /Manual/cr_restriction_enzymes.doc (1006 words)

	RE Resource, Chapter 2.1
		However, digests using multiple enzymes that have different buffer requirements may demand the use of alternative buffers and may require adjustments in the number of units of enzyme used.
		Restriction enzyme activities are relatively insensitive to the Mg concentration; similar rates are observed from 5-30mM.
		An analytical restriction enzyme reaction is usually performed in a volume of approximately 20µl on 0.2-1.5µg of substrate DNA using a 2- to 10-fold excess of enzyme over DNA, based on unit definition.
	www.promega.com /guides/re_guide/chaptwo/2_1.htm (1658 words)

	FAQs for Restriction Endonucleases, New England Biolabs
		If further manipulations of the digested DNA are required, heat inactivation (raising the temperature to 65 or 80°C for 20 minutes) is the simplest method of stopping a reaction.
		For each restriction enzyme, we report the minimum number of units (1.0, 0.5, 0.25 or 0.13) required to digest 1 µg of substrate DNA in 16 hours.
		Note that DNA substrates are digested at varying rates, the actual number of units required for a complete digestion will change from substrate to substrate.
	www.neb.com /nebecomm/products/faqcategory1.asp (1219 words)

	Lab #1, MOLB4170
		One unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1 mg of substrate DNA (usually lambda phage DNA).
		Restriction digests are typically incubated at 37 for one hour or more.
		Some restriction endonucleases are contaminated with nonspecific exonucleases and, as a consequence, the DNA in the restriction digest will become degraded during longer periods of incubation.
	w3.uwyo.edu /~uwmbio/molb4170/lab1.html (935 words)

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		Each restriction enzyme has a set of optimal reaction conditions, which are given on the information sheet and in the catalogues supplied by the manufacturer.
		In general, digestion for longer periods of time or with excess enzyme does not cause problems unless there is contamination with nucleases.
		When digesting multiple DNA samples with the same enzyme, calculate the total amount of enzyme that is needed.
	www.biology.ucsd.edu /classes/bicd101.SP06/Restrictiondigest.doc (1195 words)

	Restriction Enzyme Digest and Gel Electrophoresis Field Trip--Background
		In the Restriction Enzyme Digest and Gel Electrophoresis of DNA field trip, the students cut lambda DNA with 4 different restriction enzymes and use gel electrophoresis to visualize the DNA.
		Different restriction enzymes (and there are hundreds) recognize and cut different DNA sequences.
		The reaction is incubated at the enzyme's optimum temperature to digest the DNA.
	www.btci.org /k12/bft/red_background.html (834 words)

	Support, restriction enzymes: Fermentas Guide for Successful Digestions
		Restriction enzyme DpnI was used to digest DNA containing unmethylated targets.
		Digest your DNA under the specific conditions indicated in the product’s Certificate of Analysis (supplied with each enzyme).
		Perform digestion at the optimal temperature; refer Table "Activity of Mesophilic and Thermophilic Enzymes at 37°C" for data on the activity of thermophilic enzymes at 37°C. Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the resulting increase in salt concentration may reduce enzyme activity.
	www.fermentas.com /techinfo/re/troubleshoot.htm (1153 words)

	General guidelines for carrying out restriction digests:
		Typically, 5 ul of the restriction digest is added to 2-3 ul of agarose gel loading dyes and the entire amount of DNA + dyes is loaded.
		Be sure to run out “uncut” DNA from the same source to ensure that your restriction digestion reaction worked.
		Run your digests in the gel alongside one lane of a DNA size ladder so as to identify the approximate sizes of your restriction fragments.
	web.bvu.edu /faculty/lenzmeier/restrictiondigest.htm (538 words)

	Restriction Mapping and Analysis
		In addition, the restriction analysis data can be organised so that you can instantly focus on the particular information you're seeking.
		On the current entry level PC (Pentium III 450), the restriction analysis algorithm is so fast that for sequences less than 50,000 bases, the digestion patterns of 70 of the most common restriction enzymes is automatically determined.
		By default, the restriction analysis data is sorted alphabetically by enzyme name.
	genamics.com /expression/digestor.htm (433 words)

	restriction pattern analysis
		Restriction endonuclease digestion of DNA has been extremely useful in the characterization of these molecules since the DNA can be broken down to manageable sizes using them.
		An agarose gel of this restriction digest shows the following limit fragments: A limit fragment is what is generated when the DNA is cut at all its EcoRI sites.
		Diagram a gel of the restriction fragments that would arise from a double digest using TaqI/EcoRI of the Virus B DNA.
	www.udel.edu /Biology/fschmieg/411restriction.htm (1198 words)

	Walker's All Everything Page
		In the restriction enzyme digest the plasmid was cut at specific base pair locations with two enzymes.
		The process of DNA fingerprinting involves using restriction enzymes to cut a plasmid at specific locations so it can be differentiated from other plasmids and DNA under the same digest in an electrophoresis gel.
		The plasmid DNA that had been digested was mixed with 2-3 µl of loading buffer and centrifuged.
	www.msu.edu /~folandwa/LBS145.html (1515 words)

	Restriction Digestion Tubes (Site not responding. Last check: 2007-11-04)
		Here are the ingredients involved in a Restriction Digest, before they are all mixed together.
		In a Restriction Digest like this one, the tube on the left (DNA) would be the purified, concentrated DNA; the center tube (B) holds the Buffer; and the last tube (E) is the Restriction Enzyme.
		A typical Restriction Digest is in a final volume of 10 or 20 microliters (usually denoted as µl).
	www.life.uiuc.edu /molbio/geldigest/tubes.html (237 words)

	RESTRICTION DIGESTS
		To digest DNA with a given restriction enzyme, DNA is combined with the enzyme in a buffer that is appropriate for optimal enzyme performance.
		The ability of the enzyme to digest the DNA is assayed by gel electrophoresis.
		This concentration is usually greater than is needed to digest your DNA fragment but for your first tests, use this concentration.
	fp.bio.utk.edu /Mycology/Techniques/MT-restriction_digests.htm (819 words)

	Restriction Digest
		Restriction Digest cleaves a DNA sequence in a virtual restriction digest, with one, two, or three restriction enzymes.
		The resulting fragments are sorted by size, and they are given a title specifying their length, their position in the original sequence, and the enzyme sites that produced them.
		Use Restriction Digest to determine the fragment sizes you will see when you perform a digest in the lab.
	members.tripod.com /xiconet0/sms2/rest_digest.html (122 words)

	problem set 3
		By comparing the Bam HI digest results to the Bam HI Eco RI double digest results we see that the 7.3kb Bam HI fragment is split into a 6.7kb and 0.6kb fragment by Eco RI.
		Perhaps partial digestion with Bam HI produces a DNA fragment with an intact copy of the TrpE gene (for an example of this phenomenon, refer to the figure associated with the answer to question 10a and substitute the TrpE gene for the Drosophila gene of interest).
		This ensures that fragments that might otherwise be too small in a complete digest, or fragments that would be too large in a complete (or partial) digest with an enzyme cutting less frequently, are represented in the library.
	faculty.washington.edu /pallanck/spring.2002/probset4ans.html (2238 words)

	Teachers Guide: Restriction Enzyme Digest Analysis
		One very new and interesting application of recombinant DNA technology is in the area of forensic science, in which scientists use restriction fragment length polymorphism (RFLP) analysis to help solve rape, murder, and paternity cases.
		What they will actually be doing is analyzing the digestion fragments of the same DNA sample (lambda phage) using 3 different restriction enzymes (EcoRI, BamHI, and HindIII).
		It is anticipated that, prior to this activity, the students will have a basic understanding of DNA structure, restriction enzyme function, and gel electrophoresis as well as a cursory introduction to restriction fragment length polymorphisms (RFLPs).
	www.sci-ed-ga.org /modules/dna/resen.html (393 words)

	BI327 - Lab Protocol
		Other digest will be used to prepare the plasmid and genomic DNA for cloning.
		One unit of enzyme is usually defined as the amount of enzyme required to digest 1 ug of DNA to completion in 1 hour in the recommended buffer and temperature.
		When carrying out restriction enzyme digestions, prepare the reaction mixture up to the point here all reagents except the enzyme have been added and mixed.
	www.facstaff.bucknell.edu /pizzorno/327-lab4.html (1239 words)

	Restriction
		This step is one of the easiest and most satisfying if it works, because at the end of the digestion the students will have data they can analyze.
		Prepare a cocktail of buffer, water and restriction enzyme for the reactions.
		Note the faint band that is around 300 bp in length in all the digests.
	grows.ups.edu /description/restriction.htm (339 words)

	Restriction enzyme - Wikipedia, the free encyclopedia
		This is an example of restriction mapping, see the article on restriction maps
		While recognition sequences vary widely, many of them are palindromic; that is, the sequence on one strand reads the same in the opposite direction on the complementary strand.
		However, if either one of the adenines is methylated (a signal that the DNA is host DNA) then the enzyme acts as a maintenance methylase and methylates the other adenine.
	en.wikipedia.org /wiki/Restriction_enzyme (1214 words)

	Restriction digest - OpenWetWare
		Restriction digest involves the cutting of DNA at specific recognition sequences by enzymes.
		Tom was once having some trouble with failed restriction digests.
		Therefore, the Knight lab typically does digests in either 50 μL or 100 μL volumes rather than the 20 μL volume that the Endy lab uses.
	openwetware.org /wiki/Restriction_digest (187 words)

	Easy Subcloning
		For double digests: you almost never have to digest with one enzyme, adjust the buffer, and digest with the second.
		People who complain that blunt end ligations or triple ligations don't work well are generally inexperienced cloners who are trying to place the blame that really lies with other mistakes they made in the procedure; in my hands these ligations work ~100% of the time.
		Because the patterns from partial digests can be complex, it is often necessary to run quite a long agarose gel to resolve all the products.
	info.med.yale.edu /mbb/koelle/protocols/protocol_subcloning.html (2060 words)

	Restriction of Lambda DNA (Site not responding. Last check: 2007-11-04)
		When different restriction enzymes are used to cut a single strand of DNA such as the Lambda DNA, fragments of varying sizes are produced.
		The restriction enzymes used in this investigation are EcoR1, BamH1 and HindIII.
		Notice that the map contains the location of the gene, the position of the origin (which is the spot at which replication begins), the names of the restriction enzymes, and their restriction sites designated by the base pair distance.
	education.llnl.gov /bep/science/10/sLamb.html (1805 words)

	Genetics Glossary QR (Site not responding. Last check: 2007-11-04)
		An endonuclease that will recognize a specific target nucleotide sequence in DNA and break the DNA chain at the target; a variety of these enzymes are known, and they are extensively used in genetic engineering.
		Often due to the presence of a restriction enzyme cleavage site at one place in the genome in one individual and the absence of that specific site in another individual.
		A technique in which DNA restriction fragment length polymorphisms (RFLPs) are used as reference markers for mapping in relation to known genes or other RFLP loci.
	helios.bto.ed.ac.uk /bto/glossary/qr.htm (1889 words)

	Restriction Digests
		If a buffer can be found which allows both enzyme to work >=75% of their maximum activity then the digest can be performed simultaneously in the compatible buffer.
		If no compatible buffer can be identified then the digests must be done consecutively.
		This entails; performing the first digestion in the prescribed buffer for the enzyme; ethanol precipitating the DNA (see protocol), resuspending the digested DNA in sterile water and performing the second digestion in the prescribed buffer for that enzyme.
	www.cf.ac.uk /biosi/staff/kille/Methods/Gene/Restriction_Digests.html (251 words)

	restriction digest (Site not responding. Last check: 2007-11-04)
		Then resuspend the DNA and digest with the second enzyme in its appropriate buffer.
		The minimum number of extra base-pairs needed at the end for the restriction enzyme to work varies depending on the restriction enzyme used.
		f) The unit (U) of restriction enzyme used is usually defined as the amount of enzyme that can digest 1 ug of lambda DNA in 1 hour.
	www.biochem.ucl.ac.uk /bsm/nmr/protocols/protocols/digest.html (607 words)

	restriction enzyme-Indexed by Topic
		Restriction enzyme specificity for thymine and DNA - are thymine/uracil and DNA/RNA interchangeable?
		restriction digestion of genomic poplar dna - digestion problems with genomic dna of poplar (reply: 3)
		Proteinase digestion of tissue for DNA extraction - (reply: 3)
	www.protocol-online.org /biology-forums/restriction-enzyme.html (2085 words)

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