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. 2011 Jun 2;6(6):e20556. doi: 10.1371/journal.pone.0020556

Figure 3. Strategy for amplification and QC cloning of immunoglobulin fragments.

Figure 3

(A) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). (B) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme PstI. (C) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).