Which are the best degenrative primers to amplify heavy and light chain variable regions in an hybridoma mRNA extracted sample, to clone variable regions of the antibody?
Rather than using degenerate primers, I would suggest to use primers from regions that flank HVR of heavy and light chains. Make sure your hybridoma is from a single fusion cell to prepare DNA template. You can prepare this by limiting dilution. Once you PCR them up, clone it in plasmids and go for sequencing to get the HVR sequence.
thanks Goodwin, can you please explain for me where exactly this flank regions. I will appreciate your answer.
Flanking regions are the DNA sequences 5' and 3' sides of HVR sequence which is not a variable region. I guess that's what you asked me right?
Syd Labs, Inc (sydlabs.com) provides the service to clone and sequence antibody VH and VL.
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