Another popular screening approach is positive selection, whereby a gene lethal to the bacterial host is located in the MCS of the vector. Successful ligation of an insert into the lethal gene in the MCS prevents its expression, allowing only transformed cells with insert-carrying vectors to survive. One basic approach is to perform restriction digestion of the vector extracted from the positive or white colonies and examine the resulting banding patterns from gel electrophoresis.
Restriction enzymes must be carefully chosen and can be used to confirm the size and orientation of the insert. This method requires PCR primers that are specific to the insert, to the flanking vector sequences, or both, to detect the insert.
To determine the orientation of the insert, a set of primers that can detect the vector and the insert in a single reaction can be designed Figure 8. App note: Colony PCR. The most definitive way to identify the insert is Sanger sequencing also known as dideoxy sequencing.
While this is a good way to confirm the presence and precise sequence of the insert, this approach may be time-consuming and cost-prohibitive, depending upon the number of colonies to be screened. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. Traditional Cloning Basics. See Navigation. A general workflow for traditional cloning includes the following steps Figure 1 : 1.
Vector preparation 2. Insert preparation 3. Ligation 4. Transformation 5. Colony screening. Figure 1. Traditional cloning workflow.
Figure 2. Figure 3. Common restriction enzyme cloning strategies. A Double digestion of the vector and the insert e. B Single digestion of the vector and the insert with two separate restriction enzymes e. C Single digestion of the vector and the insert with the same restriction enzyme e. D Single digestion of the vector and the insert with two restriction enzymes with compatible ends e. Figure 4. Enzymatic digestion to produce blunt ends from overhangs created by restriction digestion.
Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team. Login processing This is a sample clip. Sign in or start your free trial. Previous Video Next Video. Overview Procedure. Overview In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. Log in or Start trial to access full content. Examination of the Telomere G-overhang Structure in Trypanosoma brucei.
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If you need immediate assistance, please email us at subscriptions jove. Thank You. Please enjoy a free hour trial. In order to begin, please login. Please click here to activate your free hour trial. If you do not wish to begin your trial now, you can log back into JoVE at any time to begin. Save to playlist. Filter by:. TA cloning using standard Taq DNA Polymerase may result in point mutations in the amplicon during PCR amplification of the desired amplicon and further required specific sequences to create overhangs that would facilitate cloning procedures.
Another technique called Gateway recombinational cloning requires DNA recombination to transfer DNA between donor and destination vectors, but this requires additional sequences for recombination. CloneEZ kits use sticky ends in the vector and insert for cloning but linearization of vector by restriction digestion is required. Our approach neither requires any selection marker nor is there any requirement for modification of pHW vector. Our LREI cloning technique is based on exponential amplification of a megaprimer and the targeted vector, which results in a greater number of positive colonies after transformation compared to the conventional cloning strategies.
This technique is specific and highly efficient in generation of cloned plasmids, which are otherwise difficult to clone. LREI cloning increases the chances of formation of recombinant clones and growth of recombinant bacteria at lower temperatures alleviates the problem of genetic recombination, albeit at the cost of plasmid yield. In summary, this technique can be applied to clone all influenza gene segments using universal primers, which would help in rapid generation of influenza viruses and make the study of influenza virus biology easier.
Further, the problem of genetic instability in bacteria encountered in influenza gene segments can be alleviated by growing recombinant bacterial cultures at a lower temperature.
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