Hi all, I am planning to generate an immunized mouse Ab phage library. I have mice immunized and will collect pan B cells and get RNA and CDNA. The key question is which primer set i should use next. I see the Current Protocols in Immunology (2002) 10.19B.1-10.19B.31 listed the detail primers. Also I see primers listed in Antibody Engineering Vol. 1, Chapter 3 . I find company such like PROGEN sells Mouse IgG Library Primer Set. Could you suggest which set of mice primer is better chocie ? Or if you have your own primers. I appreciate your help!
'Construction of a Large Naïve Human Phage-Displayed Fab Library Through One-Step Cloning' Ref https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423197/ includes a table of Oligonucleotide primers used for construction of the library
Hi Caroline, thank you for your attention. But I am looking for primers to amplify mice Ab. the reference in the paper you mentioned used primers for human library. I know most people are working on human library, not mouse library. In my case, it might be hard to screen human Ab library as most of them are naive Ab library.
OK - this time the human version is available before mouse...so select the GENES of human (clearly selected for the range they cover) and ID the mouse homolog to find suitable primers !
Schaefer, J. V., Honegger, A., and Plückthun, A. (2010) in Antibody Engineering: Construction of scFv fragments from hybridoma or spleen cells by PCR assembly (Kontermann, R., and Dübel, S., eds) Vol. Vol. 1, 2nd edit., pp. 21-44, Springer-Verlag, Berlin Heidelberg, Germany. (http://www.bioc.uzh.ch/plueckthun/pdf/APpub0307.pdf) is better than Burmester, J., and Plückthun, A. (2001) in Antibody Engineering: Construction of scFv fragments from hybridoma or spleen cells by PCR assembly (Kontermann, R., and Dübel, S., eds), pp. 19-40, Springer-Verlag, Berlin Heidelberg, as it both takes into account the improved sequence database and the practical experience with the older dataset.
Hi Annemarie, thank you for your suggestion. Actually I am looking at the Schaefer, J. V's publication which is your paper as well. I notice your primers covering both kappa and lamda chain which is exactly what i want. May I ask you one more question about the RT-PCR? I am willing to use primers from constant regions of heavy chain, K and L chain. Would you share the sequence for these primers? Such like a universal CH primers covering all isotypes of IgG.
Hi Annemarie again. I read your paper carefully and I am learning a lot from it. So you don't use C region reverse primer for RT-PCR, you only use the random hexamer primer to get the first strand cDNA. Then you use the first-strand cDNA reaction as template to amplify V region fragments. Is my understanding correct? By doing this, it can save a lot time and effort. Because if I design 5 reverse primers for RT-PCR ( C region of IgG1, IgG2a, IgG2b, IgG2C, IgG3, k and L. I wouldn't dare to mix these 5 primers for heavy chain because anyone of them may not work without knowing), that is 5 reactions. Can you comment how successful your method is?
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