I am trying to generate monoclonal antibody by phage display method. After mRNA to cDNA conversion, how many numbers of cDNA from the cDNA pool to be cloned into phagemid vector. What determines the number of cDNA to be cloned into phagemid vector or is it just a random number. Because the cDNA pool contains many variant sequences, how can we make sure that we include all the variant sequences to be cloned into the phagemid vector. Thank you in advance.
You design the primers in such a way that you assemble your PCR products in such a way that you integrate a single VL and VH gene in the correct orientation into each phagmid vector to generate e.g. a Signal-sequence - Flag-tag- VL-linker-VH-linker-gene III fusion protein. See fig. 3.1 in https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0307.pdf and https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0113.pdf for more detailed explanations of the reasons behind particular design choices. The diversity of your phage library is limited by the transfection efficiency you achieve when you transfect the phagmid into the bacteria.
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