A pediatric anti-HIV-1 subtype C scFv recombinant phage library was constructed as described previously (43, 44, 46, 47) with a few modifications. Briefly, one million PBMCs from each of the nine select pediatric cross-neutralizers were pooled and total RNA was isolated by Trizol reagent (Sigma) and then reverse transcribed to cDNA, using the Reverse aid M-MuLV reverse transcriptase (Thermo). For construction of scFv, the heavy chain and light chain variable region genes were amplified using specific primers (IDT) (Table S2 in Supplementary Material) (46) and hot start Taq DNA polymerase (Fermentas, USA). An equimolar mixture of pooled heavy and light chain DNA was used in the second round of assembly PCR using Pfu DNA polymerase. Full length scFvs were amplified by pull-through PCR reaction using Hot start Taq DNA polymerase and forward primer PTFw 5′ CCT TTC TAT GCG GCC CAG CCG GCC ATG GCC 3′ and reverse primers PAK kappa Sfi 5′ TCA GCA TGG CCC CCG AGG CCG CAC GTT TRA T 3′, and PAK lambda Sfi 5′ TCA GCA TGG CCC CCG AGG CCG CAC CTA RRA C 3′ (R = G and A). The scFvs were resolved on agarose gel and purified using gel extraction kit (Qiagen). The library was constructed by ligating the scFv into pAK100 phagemid vector by using T4 DNA ligase (New England Biolabs) followed by transformation into TG1 electrocompetent cells by electroporation (current 25°F, resistance 200 Ohms, voltage 2,500 V) (BioRad). The transformed cells were plated on to 2XYT medium agar plates containing chloramphenicol (30 µg/ml) and incubated overnight at 37°C. A glycerol stock of the recombinant scFv library was made and aliquots stored at −80°C.