TA cloning employs a thermostable Taq DNA polymerase capable of amplifying short DNA sequences. Their choice depends upon the nature of the vector and the type of PCR enzymes used in cloning. TA cloning and blunt-end cloning represent two of the simplest PCR cloning methods. For PCR optimization, reaction component concentrations, annealing temperatures, and template amounts are of importance. Ligation requires that either the insert or vector has 5′-phosphorylated termini therefore, if the cloning vector lacks 5′-phosphorylated ends, 5′-phosphate groups must be added to the PCR primers during synthesis or by T4 polynucleotide kinase for successful ligation. Primer design tools are available to bioinformatically evaluate and select suitable target-specific primer sequences for amplification. With respect to PCR amplification of a sequence of interest, primers must be designed and PCR conditions ( components and cycling) optimized for efficient and specific amplification of the template. Nevertheless, there are a number of considerations related to: PCR primers and amplification conditions, the cloning method of choice and the cloning vectors used, and, finally, confirmation of successful cloning and transformation. Furthermore, PCR cloning provides a simpler workflow by circumventing the requirement of suitably-located restriction sites and their compatibility between the vector and insert. ![]() With PCR amplification, this cloning technique requires much less starting template materials which include cDNA, genomic DNA, or another insert-carrying plasmid (see subcloning basics). PCR cloning offers some advantages over traditional cloning which relies on digesting double-stranded DNA inserts with restriction enzymes to create compatible ends, purifying and isolating sufficient amounts, and ligating into a similarly treated vector of choice (see insert preparation). PCR cloning is a method in which double-stranded DNA fragments amplified by PCR are ligated directly into a vector.
0 Comments
Leave a Reply. |