I understand that university will upload softcopy of my final year project in pdf. Isolate and purify phage genomic dna overview there are various ways to extract clean dna from a high titer phage lysate. A one step miniprep for the isolation of plasmid dna and lambda phage particles george lezin1, yasuhiro kosaka1, h. The following day they begin the protocol at step 1. A new and fast method for prearing high quality lambda dna. Purify recombinant dna plasmids from overnight culture. The lysates are dirty in that they contain spent media components, cell wall debris, flagella, nucleic acids, bacterial proteins and unassembled phage proteins in addition to the phage themselves. One reason is the limited knowledge on phagehost interaction in the intestine 15. Using the nanodrop to quantify dna or phage objective to quantify the amount of dna in a phage or genomic dna sample. Pdf isolation of bacteriophages from environmental sources. Its very high rate of infectivity makes it ideal for creating large numbers of clones. Phage genomic dna extraction modified promega wizard method. The most commonly used procedures for lambda phage dna isolation include purification of intact phage particles by chromatography 34 or csci gradient centrifugation,5 and purification of dna by.
Although the procedure was developed for lambda, it works for just about any phage. We will be purifying the dna from our recently isolated viruses using a procedure developed for purifying lambda dna. For phage plating, it is important to use solid media that is optically clear in order to be able to visualize plaques against the bacterial lawn. Isolation of escherichia coli bacteriophages from the stool. The addition of small positively charged molecules such as putrescine. The classical large scale isolation of lambda dna is based on the concentration of the phage by peg precipitation. A onestep miniprep for the isolation of plasmid dna and. Aug 15, 2011 here, we have described a onestep, onesolution inexpensive and timeefficient nidbased approach to isolate high quality plasmid dna and lambda phage particles. Isolation and purification of bacteriophages how do you isolate a bacteriophage phage and obtain a pure phage preparation. Addition of carrier dna may also be necessary during dna isolation when the expected yield of dna is low. An improved phage dna isolation method has been reported by using zinc chloride as a phageprecipi tation agent 7.
We go on to discuss the various methods available for purifying phages as well as considerations of the degree of purification which is sufficient for various applications. Isolation of escherichia coli bacteriophages from the. This article contains protocols for the isolation of phages from environmental. Components sigmas lambda dna ecor i digest is provided in a solution of 10 mm trishcl ph 8. Lambda phage is a temperate bacteriophage that infects escherichia coli it have alternative replication pathways. Basic microbiology techniques 06112011 phage enrichments 07122011 subculturing phage isolates 07122011 plating out phage 04182011 making a plate lysate 07122011. Nidbased plasmid dna isolation procedures have been used in a. Mar 11, 2015 the phage titre is also important for reaching a productive infection during phage therapy, but the titre of individual phages would in a phage cocktail get lower and lower as more phages are added.
Different restriction enzymes recognise specific sequences of bases in this dna and cut it at precise. Phage lambda dna is a common substrate for restriction endonucleases and for generating dna size marker fragments. A onestep miniprep for the isolation of plasmid dna and lambda. The large amounts of high quality dna needed for use as vectors or for markers for gel electrophoresis can be obtained in this way. The dna sequence used to construct a phage alpha restriction map includes these mutations. Phage lambda dna ecori digest readytouse geneonbioscience. Virus particles may need to be concentrated before dna isolation by ultracentrifugation, ultrafiltration, or precipitation. Isolation of bacteriophages from environmental sources, and creation and functional screening of phage dna libraries chapter pdf available october 20 with 5,896 reads how we measure reads.
These fascinatingly simple and diverse entities are also valuable as diagnostic tools, genetic screening vectors, and potential therapeutics. This one requires 1 ml of sample, and utilizes a commercial dna cleanup kit. Protocols from sullivan lab updates and discussions best. Phage lambda eco ri is ideal for qualitative and quantitative analysis of dsdna availibility of sample size may be limited. Historically, phage therapy was largely abandoned in the west in the 1940s due to the advent of chemical antibiotics, and the unreliability of phage based treatments when compared to antibiotics. Integrated viral dna is prepared using the same procedure as for isolation of genomic dna from the relevant sample.
The original protocol on which this is based was devised by greg freyer and published by cold spring harbors dna learning center. Rapid restriction mapping of dna cloned in lambda phage. Thus, while chloroform is only active with specific genotypes, nids are effective irrespectively of the phage genotype. Three samples of lambda p hage dna are incubated at 37 degrees c. The phage titre is also important for reaching a productive infection during phage therapy, but the titre of individual phages would in a phage cocktail get lower and lower as more phages are added. The bacteriophage lambda has doublestranded dna which is 48 502 basepairs in length. Traditional phage dna extraction methods are tedious and time consuming. A phage with narrow host range andor low efficacy must.
Tan gim cheong supervisor from the department of biomedical science, faculty of science. Current phage dna extraction procedures usually require a purification step because dna isolation from complete cell lysates leads to heavy contamination of phage particles with cell debris. For ligation of the iac dna into the lambda dna, it is important that the lambda dna is used in considerable excess of the iac dna to avoid gross generation of concatemeric inserts. She happened to observe streaks of mixtures of two types of e. Ten ul of the ladder should be diluted in gel loading buffer and then loaded in a single lane on an agarose or polyacrylamide gel. Phage lambda eco ri is ideal for qualitative and quantitative analysis of dsdna availibility of sample size may be limited phage lambda dna ecori digest readytouse geneonbioscience english pages. Add proteinase k to 100 gml and incubate at 65c for 45 min. This is done most easily by phenol extraction method a. The isolation of phage dna from a purified phage preparation simply. Protocols from sullivan lab updates and discussions best at. A onestep miniprep for the isolation of plasmid dna and lambda phage particles article pdf available in plos one 68.
One reason is the limited knowledge on phage host interaction in the intestine 15. Preparation of phage lambda dna lambda biolabprotocols. Hi all, i want to isolate the t4 phage dna genome and then use it as template for pcr. A miniprep is a protocol for isolating plasmids from bacterial cultures. Sambrook j, fritsch ef, maniatis t 1989 molecular cloning. Save 50100 l of the phage as a high titer stock for future endeavors.
Sigmas lambda dna ecor i digest contains 6 fragments from 3,530 21,226 bp. Phage lambda dna ecori is ideal for qualitative and quantitative analysis of dsdna. I attach you the protocol we use with the lambda kit modifications for lab. This is achieved by plating a phage suspension using the double agar method, and a susceptible host strain, to obtain plaques and further. Minipreps are used to isolate small quantities of dna from bacterial colonies to screen colonies for the correct dna. If the condensed state of the phage dna is stabilized, ejection of the dna does not occur. The nid method extracted about 50% less lambda dna compared to full cell lysis figure 4, lanes 56.
However, a protocol for the restriction mapping of a clones by partial digestion of dna labelled at one end by dna polymerase i has been developed by clark et al. Phage research has resulted in important strides in biology, from the elucidation of the function of dna and the discovery of messenger rna to the study of basic molecular interactions and genetic regulation. Phage genomic dna extraction modified promega wizard. For large scale isolation of phage dna, ci857sam7, a mutant carrying four known mutations, is used. Microscopic analysis showed that the nid procedure fragments li. Kuehn3, luca brunelli1 1division of neonatology, department of pediatrics, the university of utah school of medicine, salt lake city, utah, united states of america, 2department of. To the remainder, make the solution 10 mm excess edta, 0. In this protocol you extract the genomic dna from the phages in a lysate. The 48 kb dna fragment of lambda phage is not essential for productive infection and can be replaced by foreign dna. We also provide an optimized protocol for the creation and functional screening of phage dna. A new method for purifying lambda dna from phage lysates dna. A phage with narrow host range andor low efficacy must thus be discarded when assembling a phage cocktail.
Internal control dna for pcr assays introduced into lambda. Isolation, culture, and characterization of bacteriophages. Phage genomic dna can be extracted from phage lysates using proteins and dnas inherent chemical properties, in a. In 1950, esther lederberg, an american microbiologist, was performing experiments on e. This is achieved by plating a phage suspension using the double agar method, and a susceptible host strain, to obtain plaques and further purifying the phage contained within the plaque.
Isolation of singlestranded dna from m phage using. A new method for purifying lambda dna from phage lysates. Additional culture tubes and ce ntrifuge tubes wi ll be required. Oct 15, 2018 training video for performing phage dna extraction. To reproduce, bacteriophages or phages must take over the molecular machinery of their bacterial hosts. The choice of phage strain and the methods of phage preparation are now thought to have been critical to the success or failure of phage therapy trials.
The dna can be run on an agarose gel to visualize the dna or can be subjected to. Lambda dna is a common dna substrate extracted from bacteriophage lambda ci857ind 1 sam 7 and stored in 10 mm trishcl ph 8. Large numbers of clones are often needed for construction of gene libraries that represent large populations of molecules. Pdf isolation of bacteriophages from environmental sources, and. Laboratory manual, cold spring harbor laboratory, new york, 2. Historically, phage therapy was largely abandoned in the west in the 1940s due to the advent of chemical antibiotics, and the unreliability of phagebased treatments when compared to antibiotics. Isolation and characterization of lambda phages carrying.
Lambda phage dna, methylated from escherichia coli host. Both lysozyme and tween 80 were critical for phage isolation as in the absence of either one, extraction efficiency decreased markedly compared to the buffer containing all components figure 4, lanes 35. A very common technique in molecular biolog y is commonly referred to as minipreps, which usually use an alkaline lysis method. What is the best method for the isolation of bacteriophage dna. Purification of phage nucleic acid lambda contains doublestranded dna containing 48,502 bp. Dna restriction analysis in this experiment, dna from the bacteriophage lambda 4 8,502 base pairs in length is cut with a variety of restriction enzymes and the resulting fragments are separated using gel electrophoresis. History of lambda phagein 1950, esther lederberg, an american microbiologist, was performing experiments on e. Training video for performing phage dna extraction. Here, we have described a onestep, onesolution inexpensive and timeefficient nidbased approach to isolate high quality plasmid dna and lambda phage particles. Lambda bacteriophage of e coli is a well known doublestranded. Next the l dna is ejected from the phage and taken up by the bacterium. The large amounts of high quality dna needed for use as vectors or for markers for gel electrophoresis can be. Jul 14, 2014 lambda phage is a temperate bacteriophage that infects escherichia coliit have alternative replication pathways.
Isolation of singlestranded dna from m phage using qiagen. Pdf a onestep miniprep for the isolation of plasmid dna. Background nucleic acids absorb light at a wavelength of 260 nm. Lambda dna ecor i digest for dna electrophoresis sigmaaldrich. Jul 12, 2011 phage genomic dna extraction modified promega wizard method.
A one step miniprep for the isolation of plasmid dna and. Wurtzel, department of biological sciences, lehman college lambda phage is a commonly used vector for transgenes. Phage choice, isolation, and preparation for phage therapy. The most commonly used procedures for lambda phage dna isolation include purification of intact phage particles by chromatography 34 or csci gradient centrifugation,5 and purification of dna by chromatography. Molecular biologyphage protocols protocol online your. It is thought that this bypassing of lambda immunity is due to the. In contrast to these direct labelling techniques, indirect end labelling is inherently asymmetric, removing the need for a recleavage step. Lambda dna ecor i digest for dna electrophoresis sigma. This methylated lambda dna is not completely digested by bcl i, cla i, mbo i, mbo ii, taq i or xba i whereas nonmethylated lambda dnaproduct number d3654 is only partially cleaved. This methylated lambda dna is not completely digested by bcl i, cla i, mbo i, mbo ii, taq i or xba i whereas nonmethylated lambda dna product number d3654 is only partially cleaved. Pdf isolation of bacteriophages from environmental. The protocol is based on the concentration and purification of bacteriophage particles from crude lysates using small deaecellulose columns.
If you publish using this method, please use the following citation. Basic microbiology techniques 06112011 phage enrichments 07122011 subculturing phage isolates 07122011 plating. Phage dna isolation kit product insert norgen biotek. Even the most basic data on the genetic diversity of e. H3 dna is lambda phage dna digested to completion with hindiii. Isolation of singlestranded dna from m phage using qiagen plasmid kits en. Jun 02, 2016 03 phage titering with top agar mixing top agar and culture on lb agar plates duration. Lambda phage will enter bacteria more easily than plasmids making it a useful vector that can destroy or can become part of the hosts dna. To our knowledge, this is the first time miniprep crude lysates are tested in a downstream application.
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