Nucleobond pc 2000

CMV/R expression plasmids encoding wtRBD or gRBD fused to multimerization platforms (Fig. S3A) were prepared using NucleoBond PC 2000 (TaKaRa Bio USA Inc.) and confirmed to be essentially endotoxin free using Pierce Quant chromogenic endotoxin quantification kit (Thermo Scientific) according to the manufacturers’ instructions. Female 8- to 9-week-old BALB/cJ mice were electroporated with 60 μg DNA in each hindquarter for a total dose of 120 μg on day 0 and day 14. Electroporations were conducted on a Harvard Apparatus ECM 839 BTX system in LV mode at 40 V using 8 pulses, with a pulse length of 100 ms, at 100-ms intervals with unipolar polarity. Sera were collected before the initial inoculation (preimmune sera) and on day 21. All sera were heat inactivated for 30 min at 56°C and stored at −80°C for reuse.

Oligonucleotide primers used these studies are described in S3 Table. Plasmids were purified from E. coli using QIAprep spin and midi kits (Qiagen, Valencia, CA) or NucleoBond PC2000 (TaKaRa, Mountain View, CA). Bacterial genomic DNA was extracted using the Gentra Puregene Yeast/Bacteria kit (Qiagen). Except where noted, routine cloning was performed using the In-Fusion HD Cloning Plus kit (Takara Bio USA Inc., Mountain View, CA). Routine and high-fidelity PCR amplifications were performed using RedTaq (Denville Scientific, Metuchen, NJ, United States) and CloneAmp HiFi (Takara Bio USA Inc., Mountain View, CA), respectively. DNA sequencing was performed by Genewiz, Inc. (Cambridge, MA) and analyzed using MacVector v17.0.1 (MacVector, Inc., Cary, NC, United States). Oligonucleotide primers were purchased from Sigma-Aldrich (St. Louis, MO); S4 Table provides primer sequences.