26 Aug 2011
Graduate Aptitude Test in Engineering (GATE) is an all India test. The test is carried out jointly by the Indian Institute of Science and seven Indian Institutes of Technology for admission to Masters in Technology (M.Tech) post graduate program. GATE is administered by these top technology institutes coming under the Department of Higher Education and National Coordination Board.
GATE carries out through the establishment of eight zones. The zones and the equivalent administrative institutes are:
· Zone-1: Indian Institute of Science Bangalore
· Zone-2: Indian Institute of Technology Bombay
· Zone-3: Indian Institute of Technology Delhi
· Zone-4: Indian Institute of Technology Guwahati
· Zone-5: Indian Institute of Technology Kanpur
· Zone-6: Indian Institute of Technology Kharagpur
· Zone-7: Indian Institute of Technology Madras
· Zone-8: Indian Institute of Technology Roorkee
Structure of the Graduate Aptitude Test in Engineering
The GATE test duration is 3 hours. There are 65 questions with a maximum of 100 marks. Objective type questions are included in the GATE test. In the AE and TF papers, there are 60 multiple type questions. Four alternatives will be there. Numerical questions have a number as the answer.
Geophysics, Fiber Engineering, Aerospace and Mining papers can be answered through online. Some other papers with the same pattern are Geology and Textile. Candidates can mark right alternative by darkening the bubbles for all other papers. Numerical questions do not have negative marking. All other papers follow negative marking. There is General Aptitude part that carries 15 marks. Applicants can select any one paper from the list of 21.
Computer Science and Information Technology
Computer Science And Information TechnologyEngineering Mathematics
Electronics and Communication Engineering
Electronics And Communication EngineeringEngineering Mathematics
Geology and Geophysics
List of GATE centers
Offline Examination Cities
Colleges Accepting the GATE Score
Applied Mechanics And Design
Fluid Mechanics And Thermal Sciences
Manufacturing And Industrial Engineering
Atomic And Molecular Physics
Nuclear And Particle Physics
Solid State Physics
Thermodynamics And Statistical Physics
Production and Industrial Engineering
Textile Engineering and Fiber Science
Textile Engineering and Fiber Science
The following categories of applicants are qualified to appear for GATE:
· Aspirants in the second year or more in the Integrated Master’s degree course (Post-B.Sc.) in Engineering/Technology
· Candidates with eligibilities got through tests carried out by professional societies recognized by UPSC/AICTE.
· Post graduates in Science/statistics/maths/computer application or applicants who are in the last year of these courses
· Students who are pursuing the last year of any of these courses
· The applicants who finish B.E/ B.Tech/B.Arch course
· Those who are in the third or higher year of Five-year Integrated Master’s degree course and Dual Degree course in Engineering/Technology
How to Apply?
The aspirants can collect the GATE applications forms from the banks nearby. You could also pick up application forms from from the Zonal offices of IITs and IISB. Aspirants have to fill in application forms and send along with a DD. Online application facility is also there. The cost of the form Rs.1000. Online form cost is Rs.800 for OBC and general category students. For SC/ST students the cost is half of that.
How to fill the Form?
Aspirants can fill the application forms cautiously. They must check that whether there is application number printed on the form. It is necessary to quote the Application Number in every future contact with the respective zonal GATE office. It is better to take a copy of the application form. The Application Form will be stated imperfect if the instructions are not followed. Applicants must darken the bubbles with HB pencils. It is good to follow the instructions mentioned in the form.
The GATE exam carried out in the month of February. The application forms can collected online on September 4th week. The forms are available until October 3rd week. The application forms can be collected from the GATE office counters at the end of October. Filled forms can submitted online until October last week and offline before November 1st week. The results come out by March mid week.
List of GATE centers
Offline Examination Cities
Colleges Accepting the GATE Score
Contact Address for GATE
· Address: Chairman, GATE, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016
· Phone No: 011-2659 1749
· Fax No: 011-2658 1579
· Email id: Gate@admin.Iitd.Ernet.In
Coaching Centers for GATE
· B.R.M. Academy
· Brilliant Tutorials Pvt. Ltd.
· Career Forum
· Elite Academy
· Engineering Solutions
· Engineers Institute of India
· Engineers Study Circle
· Gyan Bindu Academy
· IAS / IES Made Easy (Engineers India Centre)
· Institute of Mathematical Sciences (IMS)
· Pathfinder Academy
· Science Point
· Trump and Gates
· Vani Institute of Engineering & Technology (VIET)
The GATE test examines candidates on their fundamentals. There are mainly derivation questions. The aspirants must get their fundamentals correct, and then test on the fundamentals. The aspirants can choose best coaching material. They can ask their seniors as what they used to clear the GATE exam. Here are some constraints on which aspirant can choose whether he/she should go for a particular coaching center, use a particular coaching material, or tests:
· Material Collection - All the relevant books related the subject, Some books useful for pre-requisite knowledge on the topic, Some excellent guide books for GATE and Previous questions papers
· Keep contact with a few expert and GATE experienced individuals
· Preparations - read minimum 5 books, it will broaden applicants knowledge, note down the possible concepts, solve problems as maximum as possible, think about different tricks in solving problems, go for series of self examinations Solve more and more problems, find out more and more new tricks
How and Where To Get the Results?
The results are available in particular IIT website. The applicants can enter the seven-digit number for to get the result from the official website.
The cut off varies based on the institutes and stream of course. Some colleges are keeping a cut off 700 for Computer Science direct admission. Other streams cut off are around 750. For getting interview call, the aspirants must obtain at least 600. Top institutes demand at least 90% and above.
The GATE score is valid for two years from the year one crack the test.
14 Aug 2011
Once the collection is actually constructed it is screened for any specific gene of interest. Testing is dependant on homology between the probe and among the clones in the library. The probe will be normally the nucleic acid that has some sequence homology to the gene that's represented within the library. The collection is the collection of clones from the origin DNA which is put into a cloning vector. For example, a bean genomic lambda library consists of items of the entire complement associated with bean DNA, through 9-20 kb in size, cloned into a lambda vector. Any kind of probe accustomed to screen the actual library must have a few homology to some clone in the bean library, which homology would permit you to choose the appropriate clone from the numerous clones that don't include your own DNA associated with interest. Probes arrive in a number of forms. The more homologous (equivalent within DNA sequence) the probe is to the sequence which is being sought, the simpler it is to pick the duplicate from the collection. For example, a bean lambda library is recognized as the genomic library since it consists of all of the DNA sequences present in the actual bean genome. A bean leaf cDNA library, although, would contain simply those sequences which are expressed in the leaf once they possess undergone digesting. Therefore the actual cDNA clones wouldn't include any of the intron sequences or the managing elements of the gene. In order to obtain a genomic duplicate through the actual bean lambda library, the best probe will be a cDNA clone obtained through screening the bean cDNA library. This type of probe, the probe that contains the precise sequence of the sequence that's being searched for, is known as a homologous probe. However how was the original cDNA clone acquired so that it might be used as a probe. The actual cDNA library that the clone was obtained might have been screened with a probe through another varieties which represents coding details for that exact same gene but through an additional species. For example, the bean leaf cDNA library might be tested with a tomato RUBISCO smaller subunit clone. A probe which has DNA that encodes for the gene associated with curiosity however through an additional varieties is called a heterologous probe. Many grow genes have been cloned through screening your local library along with each homologous as well as heterologous probes.
Polymerase chain reaction methods are actually popular to duplicate genetics. To make use of this method primers should be designed which are contrasting for your target sequence. One oligonucleotide is going to be contrasting towards the anticoding strand (the DNA strand of the gene contrasting in order to the mRNA and used like a template for transcription). The second oligonucleotide is going to be contrasting towards the coding strand (the DNA strand of the gene complementary in order to the actual anticoding strand).
When the gene may be cloned within an additional varieties, you can use that sequence info to design primers in order to amplifiy a fragment of the gene in the DNA of the varieties. Often, the actual gene a person are interested in hasn't already been cloned, however, you may have isolated the actual proteins. If this can be the situation, the initial step of the method is to acquire partial protein sequence information of the protein. Micropeptide sequencers are available that can rapidly generate sequence info. From this particular sequence, you should use change translation of the amino acidity sequence to obtain the nucleic acid sequences of the amino-terminal fragment as well as a carboxy-terminal fragment. In this case the actual fragment complementary to anticoding strand will be a direct transformation from the hereditary signal for the amino acids in the amino-terminal fragment of the proteins. The strand complementary to the coding follicle is going to be contrasting to the produced nucleic acidity sequence of the carboxy-terminal fragment. Let's imagine which the next may be the N-terminal series from the peptide that you wish to derive a artificial oligonucleotide:
These would be a suitable probe for that gene.
5'-A T G T G T/G G T N A A A/G A G N C C - 3'
(D = A, T, G, as well as C)
When you're making these probes several concepts should be considered. First, the genetic code is actually created in the mRNA sequence. This particular synthetic oligonucleotide will thus, be contrasting towards the anit-coding strand that can be used since the template for the mRNA. Next, simply because you only understand the actual amino acid and never the actual DNA sequence you have to deal with redundancies of the genetic code. For example leucine can be represented through CTA, CTT, CTG, CTC. Thus your own synthetic oligonucleotide needs to contain all of the options. Therefore the over sequence is actually a combination of 64 (4x4x2x2) different sequences. Due to this redundancy it is usually best to make you oligonucleotide one lacking full length. This can assist some of the redundancy problems. In this example, the actual probe is 17 nucleotides long and also the redundancies with regard to proline tend to be eliminated. An additional approach to decreasing the redundancy issue would be to incorporate the nucleotide deoxyinosine from any placement where a high level or redundancy happens. Deoxyinosine is really a altered nucleotide that doesn't set with any kind of of the four angles discovered in DNA. Therefore, this doesn't impact the homology from the probe
The next could be the amino acidity sequence of the carboxy-terminal fragment:
The right oligonucleotide sequence from the above sequence for PCR amplification will be:
5'- G C N A C N G T A/G T G A/G T A A/G A A -3'
(Remember that the final redundant nucleotide in the alanine amino acid residue was not included. ) Amplification of DNA between both of these oligonucleotides will give you the homologous probe because it will signify the actual precise sequence from the gene for which you are looking. Once this particular probe is actually radiolabelled through nick-translation (as all probes must be radilabelled prior to library screening ), it may be used to screen a cDNA or genomic library for a clone complementary to the actual increased fragment.
DNA Sequencing With the Chain-Termination, Dideoxy Technique of Sanger
Once you have a very prospect clone, an individual may wish to sequence this to determine if it is similar to an additional gene currently sequenced or even be it unique. DNA sequencing is really a DNA replication dependent reaction. Both needs for DNA replication really are a DNA template as well as a totally free 3'-OH team. These requirements must be fulfilled for any sequencing procedure. The next steps illustrate the actual Sanger procedure, the most favored DNA sequencing procedure.
1. Even though this method initially used DNA cloned in a vector that may produce single- stranded DNA, sequencing is now regularly carried out within double-stranded plasmid or even cosmid vectors. PUC plasmids and related plasmids tend to be popular with regard to double stranded sequencing. To start with the process, the DNA is actually made single stranded by a mix of heating as well as alkaline problems.
2. A primer which has a free of charge 3'-OH group is actually annealed towards the vector. A handy primer may be the multiple cloning site from the pUC plasmids. DNA functionality will start here. 3. Four responses are performed, each that contains dATP, dGTP, TTP as well as 32P-dCTP and one dideoxy nucleotide in reduced focus. Dideoxy nucleotides have a hydrogen rather than a - OH group attached to the #3 carbon and cannot be employed for further extension of the chain. Therefore in this particular response, numerous synthesis products will finish with this nucleotide.
4. Add DNA polymerase towards the response. Nucleotides is going to be additional one at a time but the string may end when a dideoxy nucleotide is inserted. Because the dideoxy nucleotide is restricting a number of items, every closing using the same base, may end up being acquired.5. Separate the actual pieces on an acrylamide gel, create via autoradiography as well as browse the sequence.
9 Aug 2011
1. Substantial DNA (>100 kb) is ligated between two arms. Every arm finishes with a yeasttelomere so the product or service can be stabilized within the yeast cell. Interestingly, larger YACs are more stable compared to smaller types, which usually favors cloning of significant extends associated with DNA.
2. One arm contains an autonomous duplication sequence (ARS), a centromere (CEN) and aselectable marker (trp1). Another arm contains a second selectable marker (ura3).
3. Insertion involving DNA to the cloning site inactivates a mutant indicated in the vector DNA as well as red yeast colonies appear.
4. Transformants tend to be recognized because individuals red colonies which grow in a yeast cellular that is mutant with regard to trp1 along with ura3. This ensures that the cellular has received an synthetic chromosome along with both telomeres (on account of complementation from the two mutants) as well as the artificial chromosome consists of insert DNA (considering that the cell is actually red).
These are linear vectors that act as an yeast DNA; therefore they're called yeast synthetic chromosomes (YACs). A regular YAC, e. G., pYAC3, contains the subsequent functional components from yeast:
(1) An ARS sequence pro duplication,
(2) CEN4 sequence pro centromeric function,
(3) Telomeric sequences through the two topsgenetic materialo safety from exonuclease proceedings, as well as
(4) 1 or 2 selectable marker genes, viz., TRP J and URA3, (strategy akin to other vectors);
(5) SUP4, a selectable marker into that the genetic material insert is integrated; and
(6) The required sequences from E. Coli plasmid pro selection and procreationossibly within E. Coli. The actual telomeric sequence in yeast chromosomes is a 20-70 tandem repeat of the 6 base sequence 5'CCCCAA3' (its complementary sequence, 5TIGGGG3', occurs in the other strand); their is a hairpin loop formation by the terminus, which makes the actual genetic material duplex strong to exonuclease proceedings.
Vector pYAC3 is in effect a pBR322 plasmid into that the higher than described yeast sequences are already integrated. Subsequently, several YAC vectors have been constructed on the fundamental scheme of pYAC3. The YAC vector itself is propagated within E. Coli, but cloning is made in yeast.
For cloning, the actual vector is restricted with combining BarnHI and SnaBI. BamBI cleaves the vector by the junctions of the two TEL sequences using the fragment that is accustomed to circularize the vector professional procreation in E. Coli; this particular fragment is actually discarded
The enzyme SnaBI recognizes the solitary sequence 5'T ACGT A3' located in SUP4 and produces blunt-ended cleavage, so generating two arms from the YAC, all complete in a TEL sequence. The DNA place, consequently, must have blunt ends; it is integrated within SUP4 to create the linear YAC.
The actual recombinant YAC is introduced into TRP 1- URA3- mushroom tissue through protoplast transformation; transformed tissue are selected by plating them on the smallest standard: Single persons cells are able to grow on this standard using the intention of be inflicted with accurately constructed YAC containing lone missing and lone aptly arm of all DNA.
Recombinant clones are identified due to the insertional inactivation of SUP4 detected with a unadorned colour test: Recombinant colonies are white, while nonrecombinant ones are red. The TEL sequence of the vector is not the complete telomeric sequence, but it contains sufficient of this sequence to be able to support the creation of complete telomere some time ago the YAC is inside a mushroom cell.
Thus a Y AC is a ferry vector with the intention of is propagated in circular form in E. Coli and is used pro cloning in mushroom in a linear form. When a Y AC is a reduced amount of than in this area 20 kb, the centromeric function is unable to control imitation digit all through mitosis so with the intention of several copies of Y AC accumulate for every mushroom cell.
The centromeric function improves in YACs of 50 kb or more; YACs of 150 kb or more perform like regular mushroom chromosomes. YACs are the predominant vector logic used pro cloning of very generous (up to 100-1, 400 kb) genetic material segments pro mapping associated with complicated eukaryotic chromosomes YACs are reported to suffer from many problems, counting chimerism, deadly steps in Y AIR CONDITIONING store construction and low yields of Y AC slot in genetic materials.
The mushroom genes bestow not very good mushroom vectors can be changed into integrated into the host genome; this is known as stable transformation. It commonly occurs through homologous recombination linking the gene bestow in a vector (e. G. , LEU2) and using the intention of bestow in the mushroom chromosomes (Elizabeth. G., LEU2-). Rarely, the gene could be converted into inserted by a arbitrary DNA locate.
The homologous recombination might occur by regular crossing ended or it could occupy gene conversion (a nonreciprocal recombination). Vectors be inflicted with been invented pro distinguished frequency set up transformation; such vectors are introduced in mushroom cells in linear form and contain by their both tops sequences with the intention of are homologous to persons found by the target locate (the location where the gene bestow in the vector is to be integrated) in the mushroom genome.
Such vectors permit integration of one specified genetic material sequence by the desired locate in mushroom genome, i. E., they allow site-specific transformation (= integration) of genes.
Cosmids are ordinarily plasmid vectors with the intention of be inflicted with cos sites. The cos locate will be the solely requirement of Geonomics to be converted into packed in to a bacteriophage particle. Cosmids had been produced think it over how to dodge of the statement. How can you duplicate in to cosmid vectors?
1. Duplicate the Geonomics in to the vector while you might along with one kind of plasmid genetic material.
2. Expose your Geonomics in to the microbial cellular with a phage particle.
3. Propagate since plasmid genetic material.
Because bacteriophage contaminants may possibly take amongst 38 and 53 kilobytes regarding Geonomics and as generally cosmids are regarding 5 kilobytes, among 33 and 48 kb of Geonomics may possibly cloned in these vectors.
Complications connected with lambda and cosmid cloning.
1. Given with the intention of repeats happens to eukaryotic Geonomics rearrangements can take place via recombination from the repeats current in this area the Geonomics inserted in to lambda or cosmid.
8 Aug 2011
Bacteriophage Lambda Vectors
Interestingly enough, even bacteria tend to be affected by viruses! You may know that when viruses infect cellular material they have the ability to move their nucleic acid genetic materials for the inside of the host cell. There it replicates to produce up to several hundred new viruses. If we could substitute a number of of the virus-like genetics having a gene we're interested in amplifying, then we'd have another type of vector as well as the plasmids alreaady discussed. Furthermore, the actual host cellular might obligingly bundle up the replicated DNA that contains our gene in to new virus contaminants which are liberated from the cell.
The actual lambda genome is 48. 5 kbases in length and contains regarding 46 genes. The actual bacteriophage lambda is frequently used like a vector. It has a head structure that contains the actual viral DNA along with a long tail that is used in connection associated with the virus in order to E. coli. The original virus offers some genes which allows the viral DNA in order to insert itself into the microbial chromosome. These genes might be cut out as well as discarded to provide a location for the new gene to become put.
FIg. The lambda genome
Subsequent attachment of the virus to E. coli, the DNA in the head structure travels lower the hollow tail and enters the host cell. The linear, viral DNA has normally "sticky ends" consisting of a complementary sequence of 12 bases on each end of the molecule. The sticky ends from the linear DNA (the actual cos site) associate to form circular DNA. The virus codes because of its own DNA polymerase which leads to the synthesis associated with several hundred copies of viral DNA.
Fig.The phage life cycle
The DNA is synthesized within very long repetitive units that are cleaved at the cos sites in order to provide unit length viral DNA for attachment in to new particles. The bacteriophage DNA also codes for every one of the proteins needed to come up with new virus contaminants which in turn tend to be assembled within the host cell.
The newest DNA is trapped inside the newly-formed viral coat protein and the host cellular bursts, liberating the new particles. A most remarkable property from the proteins and the viral DNA is that total particles will assemble in the test tube whenever all of the reactants tend to be mixed together.
The infection spreads to adjacent cells and also the cycle is actually repetitive. When the infection will be carried out with E. coli cells which are spread over an agar surface, the infected areas look as clear dots (as well as plaques) surrounded by live cells. Bacteriophage might be recovered from the cear areas. Every plaque originates from the solitary contaminated cellular.
Fig.Phage lambda plaques on a lawn of bacteria
Virus particles are divided from cellular debris and the DNA separated. The procedure may be scaled up by infecting cells growing within liquid culture. More than 100 various lambda vectors happen to be prepared with regard to make use of in cloning. The DNA from the cloning vector should function as the correct size for it to become packaged in to virus particles (between 38 as well as 51kb).
The actual vector lambda gt10 (43. 8 kbases) comes with an EcoRI site inserted inside the cI gene (lambda repressor) and so could accept the DNA fragment associated with 7. 6 kbases before getting too large to be packaged in to virus particles.
Fig.The vectors lambda gt10 and Charon 16A
Other lambda vectors can include DNA fragments of up to 22 kbases (age. g. , the Charon set ).
To use lambda gt10, we'd cut the actual vector DNA and the DNA to be inserted along with EcoRI, incubate both collectively, seal along with ligase and incorporate the actual recombinant molecule into particles within the check tube:
Open lambda gt10 along with EcoRI as well as insert dual stranded DNA additionally cleaved with EcoRI.
Seal with ligase.
Package the DNA into contaminants along with a mix of mind as well as tail precursors.Infect E. coli with the reconstituted virus particles containing the brand new gene as well as recover the actual altered bacteriophage from infected tissue because described above.
6 Aug 2011
Characteristics involving plasmids
1. extrachromosomal spherical Geonomics molecules which are not the main bacterial genome
2. size range: 1-200 kilobytes
3. take functions advantageous to the host such as:
i. produce enzymes which break down anti-biotics or chemical toxins
ii. produce limitation and adjusting nutrients
4.Duplication is coupled to host replication in a:
i.stringent way - one (or two) plasmids produced during every circular associated with microbial duplication
ii.relaxed method - 10-200 copies of the plasmid made during each round of bacterial duplication; (this can be increased to 1000-2000 plasmids by stopping host protein synthesis and replication with the antibiotic chloramphenicol)
UnknownDNA is inserted into a plasmid DNA (or any cloning vector) by ligating the DNA into a complementary site in the plasmid DNA. These sites are generated by digesting the DNA and vector with the same restriction enzyme. (The site for the restriction enzyme that is chosen should only be represented once in the plasmid DNA. Therefore, when the plasmid is digested, a single, linear molecule would be generated.) The foreign DNA is then inserted into the plasmid by the action of the enzyme DNA ligase. The next step is to insert the ligated DNA into a bacterial cell for propagation. This is done by a technique called transformation. Bacterial cells tend to be given either Ca2Cl or Rb2Cl. This treatment generates pores in the bacterial cell wall and membrane through which the plasmid DNA enters. Although there is no size limitation to the ligation reaction, transformation efficiency is dictated by the size of the plasmid.
Impact of plasmid size on transformation effectiveness
Molecule size (kb)
% Maximum probability
The goal of the ligation reaction is to insert the foreign Geonomics into the vector. An undesirable ligation product also occurs - religated vector. One way to minimize this event is to treat the vector with phosphatase. This removes the terminal phosphate group from the restriction site of the vector and theoretically prevents religation of these two ends. In reality though, this treatment is never 100%, so a low-level associated with religation will occur. Therefore following transformation you will have two types of cells: those which contain the original plasmid and those that contain a plasmid DNA containing foreign DNA.
Plasmids are designed to distinguish the two types of transformation products. pBR322, the initial widely used vector, utilizes differential gear anti-biotic screening to tell apart the two types of transformation products. Let's say that we clone into the BamHI site of the vector. The insert Geonomics will then split the gene responsible for tetracycline resistance. But at the same time frame, the gene for ampicillin resistance is left undamaged. Transformed cells are first grown on bacterial plates containing ampicillin. This will kill all the cells that do not contain a plasmid. But we still cannot say which cells contain foreign DNA. Those cells that grew on ampicillin are then replica plated on plates with ampicillin and tetracycline. Individualstissue which grow in the presence of the Principen, but die under tetracycline selection contain plasmids which have foreign DNA inserts.
pBR322 would be a discovery for molecular biology, but the double screening procedure was time consuming and could be subject to error. In1981, a new number of plasmids were developed that permitted the identification of the foreign DNA containing cells in a single screening step. Theseare known as the pUC plasmids. As with pBR322, ampicillin resistance is used as one selectable marker. The actualsecond marker is based on insertional deactivationfrom the E. coli lacZ gene. The wild type gene can hydrolyze a specific dye [X-Gal (5-bromo-4-indoyl-B-D- galactopyranoside)] to a blue colour, and the bacterial colony is stained blue. A multiple cloning site has been put in to this gene. Thissite will accept fragments ending in a number of different restriction enzymes. Onattachment of DNAinto this site, the exerciseof the gene is eliminated and the colony appears white in color. Thereforetransformed colonies that contains plasmids with inserts can be distinguished from those with plasmids without inserts based on the color of the colony and the capability of the colony to develop on an ampicillin containing media.
cDNA Cloning (Cloning EukaryoticmRNA)
cDNA cloning isa technique for gettinga DNAcopy of the mRNAs that are expressed at a specific stage in the development of the plant. Inthis method, you can improvethe library that you will screen for those sequences withinwhich you are fascinated. The reagent requiredfor this type of cloning approach is mRNA. These mRNAs have a poly A+ tail. This buttpermits the isolation of poly A+ mRNA by using eitheroligo-dT or oligo-U columns. Total RNA is run through one of these columns under conditionswhich favor the binding of the tail to the matrix on the column. After thecolumn is extensively washed, the conditions are usuallychanged and the bound mRNA is isolated. This is the starting reagent for cDNA cloning.
Steps in cDNA Cloning
- Bind oligo-dT to the actualpoly +A tailof the mRNA.
- Add reverse transcriptase and make a Geonomicscopy of mRNA (cDNA). Erase the mRNA with alkaliand highheat. (First strandsynthesis)
- Add a C-tail to the 3' end of the cDNA with terminaltransferase.
- Add oligo-dG to the tailed-cDNA and make the actualsecond strand with reverse transcriptaseor the Klenow fragment of DNA Polymerase I.
- Add dC's to the 3' end of the double-stranded cDNA with terminaltransferase
- Add C-tailed, ds-cDNA to G-tailed, PstI cut pBR322 and anneal. (DNA ligase is not neededfor this step.)
- Transform E. coli cells.
- ChooseTetr/Amps cellular material. Most, howevernot all inserts maybe removedby PstI digestions.
Posted by biotech