Gel dna extraction kit

Oligonucleotides were designed to assemble expression cassettes containing the human IgƘ signal sequence followed by a primate CEACAM1 N-terminal domain, and finally a STREPII tag and tobacco etch virus (TEV) protease site. Templates for primate CEACAM1, IgƘ, and the STREPII tag/TEV protease site were synthesized commercially (Genscript & Integrated DNA Technologies, Piscataway, NJ, USA). Oligonucleotides were designed to amplify each fragment with approximately 20 bp of template overlap for PCR assembly as detailed in Supplementary file 1A and B. Individual PCR fragments for IgƘ, the N-terminal domain of primate CEACAM1, and the STREPII/TEV tag were generated using the Phusion (Finnzymes) high-fidelity polymerase and were subsequently gel-purified (Gel DNA Extraction Kit, Zymo Research). For assembly, 0.2 µL of each purified PCR fragment was added as template in a Phusion PCR reaction with the outermost primer set (IgK pCDNA GFP LIC F and GFP STREP II CEACAM1N R3). The assembly PCR was separated on a 1% agarose gel and the band corresponding to the correct assembly length was gel extracted and purified.

Two micrograms of plasmid was cut with the FastDigest PstI restriction enzyme (Thermo Fisher), and the 900 bp piece was gel purified using a gel DNA extraction kit (Zymo). This piece was then recirculized in a 20 µL reaction containing 60 ng of gel purified DNA and 1 µL T4 DNA Ligase (Thermo Fisher) in buffer. Cutting and recircularizing was necessary to increase efficiency of later sequencing steps for unknown reasons. The ligation product was then amplified with primers containing the appropriate overhangs for high-throughput sequencing and then gel purified. This piece was then sequenced through paired-end sequencing using custom primers on an Illumina (San Diego, CA) sequencing machine (Miseq or Nextseq).