What are you trying to do - clone scFv libraries from a specific species? In this case, you generate an alignment of all genomic antibody VH, VLlambda and kappa sequences recognize the N-terminal side, C-sequences to get the N-terminal side of the protein (from IMGT repertoire, http://www.imgt.org/IMGTrepertoire/) to generate a minimal set of primers sto amplify the V repertoire. Try to get all primers to anneal at as close to the same temperature as possible, to be able to use a high annealing temperature in PCR to avoid introducing destabilizing mutations through mismatched primers.

Add the appropriate sequences for affinity tags, linkers and assembly of the scFV from the V-domain sequences in the desired orientation (VL-linker-VH or VH-linker-VK.

For mouse, we did this here:

Chapter Construction of scFv Fragments from Hybridoma or Spleen Cell...

Thanks for you reply and i highly appreciate your gesture. i am going to clone scfv from cattle PBMCs. cattle has VH and 90% VLlambda. so it minimizes the issue. I have collected as many as possible mRNA sequences of VH and VL lambda of cattle available on NCBI data bank. Do i need to collect the genomic sequences instead of mRNA. the link you posted in the comment, Should i select the option of protein display there?

mRNAsequences are also ok, you just want to know the diversity of the nucleotide sequences (not protein), to design a comprehensive primer set. Our first set of primers https://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-11300 was bases on the then available mRNA sequences - knowing the genomic sequence of the murine Ig locus helped to refine the primer set for the newer publication (sorry - the ResearchGate link got lost in copy-pasteing, - try this one: https://www.bioc.uzh.ch/plueckthun/index.php?pid=3-1-30700)

Thanks for you guidance. I already have these articles and I have read them and they were very helpful in desgining some of my research plan. I want to ask that when you go for amplification of VH and VL then do you design forward primers on their conserved signal peptide part or the conserved part of Framework 1. What i understand is that the focus of forward primer design should be on Framework 1.

and when it comes to reverse primer then Framework 4. Please help me clear my doubts regarding this. Thank you very much.

We were going for direct assembly of scFv fragments, therefore we needed the full variable domain

I also need the full variable domain of VH and VL to construct scFv. So, this means i should focus on the conserved signal peptide sequence for primer design and leave nothing behind?

Since each V gene contains its own signal sequence, these sequences are not that well conserved (see http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VKLE ).

In addition, in designing the degenerate primer sets for the amplification of the V-domain genes from cDNA, we directly added sequence features needed for the generation of the phage display construct, so that a subsequent assembly PCR with defined primers and restriction digest yielded the fragment that could be directly inserted into the phage display vector containing the bacterial control elements and signal sequences needed for phage display. If we had first used primers directed against the leader sequence and C-domains (as one would use to just sequence the V-domains), we would have had to do a second PCR with degenerate primers within the V-domain to insert the tags, linkers and sequence overlaps necessary to insert the V-domains into the phage display vector. (see fig. 3.1 in the Schaefer paper.

thank you very much Annemarie, let me have a look at this and i shall get back to you for your kind feedback.

Dear Annemerie, Thank you very much for you previous suggestions. I am now trying the cloning using pComb3 vector system. But i am having a background of empty vector. Even though i gel purify the the cut vector band but still i am having this empty vector problem. 1/8 is the ration of empty vector: vector with insert. Can you please suggest how can i finish this background of empty vector or it is normal to have this kind of empty vector background. thanks

A background of empty vector can be due to the presence of uncut vector or due to re-ligation of empty vector due to incomplete dephosphorylation. Did you properly dephosphorylate your vector to prevent religation? Did you use a sufficient molar excess of insert over vector? See the suggestions, particularly the proper controls to use, at this site: https://www.addgene.org/protocols/dna-ligation/

Thank you very much for your reply. I dephosphorylated my vector using

Alkaline Phosphatase, Calf Intestinal from Promega. After dephophorylation of the restriction mix of vector, i run it on gel and cut the band of cut-vector and purify it. for ligation, i also make control of cut vector+ ligase. But i am having some bacground of emtpy vector. is it normal to have little background of empty vector?

It can be difficult to get baseline separation of cut vector and vector that has just been nicked in gel purification. In library design, where it is crucial to have a very low background of empty vector to get a high quality library, we often use a vector with a large stuffer insert that has to be cut out, resulting in a cut vector with incompatible ends (two different restriction sites, no re-ligation) and a large size difference between fully cut vector and vector that may only have been cut in one of the two sites site.