Total RNA was extracted from the peripheral blood lymphocytes with RNAqueous midi kit (Ambion) and the cDNA was prepared using MMLV reverse
transcriptase (Invitrogen) with the CALL002 primer (5’-GGT ACG TGC TGT TGA ACT GTT CC-3’). VHH coding sequences were amplified by PCR with primers
FR1For (5’-GAT GTG CAG CTG CAG GAG TCT GGG GGA GG-3’) and FR4Rev (5’-GGA CTA GTG CGG CCG CTG GAG ACG GTG ACC TGG GT-3’) annealing at the framework-1
and framework-4 regions flanking the CDRs of heavy chain antibodies, respectively. The PCR fragments were ligated into the phagemid vector pHEN4 ( 14 (link)
) after cutting with the restriction enzymes PstI and NotI. Ligated materials were transformed by electroporation in freshly prepared E.
coli TG1 cells, plated on 24×24 cm plates containing 2YT medium (16g tryptone, 10g yeast extract and 5g NaCl) supplemented with ampicillin and
glucose(2YT–AMP–GLU ) and incubated overnight at 37°C. The electroporation was performed with the Eporator (Eppendorf, Germany) in 1mm gap cuvettes
(Sigma) at 1700Kv. Colonies were scraped from the plates and stored at −80°C in 2YTmedium supplemented with 50% glycerol.
To establish the library size, dilution series (10-3, 10-4, 10-5, and 10-6)
were prepared from electroporated cells in a liquid medium and 100 µL
were plated onto 9 cm diameter 2YT–AMP–GLU plates and incubate overnight at 37°C.
transcriptase (Invitrogen) with the CALL002 primer (5’-GGT ACG TGC TGT TGA ACT GTT CC-3’). VHH coding sequences were amplified by PCR with primers
FR1For (5’-GAT GTG CAG CTG CAG GAG TCT GGG GGA GG-3’) and FR4Rev (5’-GGA CTA GTG CGG CCG CTG GAG ACG GTG ACC TGG GT-3’) annealing at the framework-1
and framework-4 regions flanking the CDRs of heavy chain antibodies, respectively. The PCR fragments were ligated into the phagemid vector pHEN4 ( 14 (link)
) after cutting with the restriction enzymes PstI and NotI. Ligated materials were transformed by electroporation in freshly prepared E.
coli TG1 cells, plated on 24×24 cm plates containing 2YT medium (16g tryptone, 10g yeast extract and 5g NaCl) supplemented with ampicillin and
glucose(2YT–AMP–GLU ) and incubated overnight at 37°C. The electroporation was performed with the Eporator (Eppendorf, Germany) in 1mm gap cuvettes
(Sigma) at 1700Kv. Colonies were scraped from the plates and stored at −80°C in 2YTmedium supplemented with 50% glycerol.
To establish the library size, dilution series (10-3, 10-4, 10-5, and 10-6)
were prepared from electroporated cells in a liquid medium and 100 µL
were plated onto 9 cm diameter 2YT–AMP–GLU plates and incubate overnight at 37°C.