Cloning of gBlocks

Full-length CDR3 regions for TCR-α and -β chains were determined from single cell TCR sequencing and custom synthesized into gBlocks (Integrated DNA Technologies, Coralville, Iowa USA) to be cloned into pSelect-GFP-Zeo vectors. The gBlock was cut with the restriction enzymes Sal-I and Nhe-I with 10x Cut smart buffer (NEB) and incubated for 2 hours at 37°C. Cut products were purified using the Qiagen PCR Purification Kit (Qiagen), as per the manufacturer’s instructions. Briefly, five volumes of buffer PB was added to the digested sample and added to the purification column. The column was washed with buffer PE and purified product eluted with 25µL of elution buffer. The insert was ligated to the similarly cut vector using 2x quick ligase buffer (NEB) and incubated for five to 15 minutes at room temperature. The ligated product was transformed into DH5alpha cells by a heat shock method. The ligation mix was added to 100µL of DH5alpha cells and incubated for 30 minutes on ice. The mix is then heat shocked at 42°C for 45 seconds then placed on ice for two minutes. Nine hundred microliters of S.O.C. Medium (ThermoScientific) was added to the tube prior to shaking for one hour at 250rpm at 37°C. Two hundred microliters of culture were plated on Agar plates (Fast-Media Zeo Agar; LB based agar medium supplemented with Zeocin; InvivoGen, CA, USA) and incubated overnight at 37°C.

Six to eight colonies were picked and cultured individually overnight in 3mL of Fast Media Zeo TB (TB-based liquid medium supplemented with Zeocin; InvivoGen) at 37°C and 250rpm. Plasmid DNA was extracted using the Wizard Plus SV Minipreps DNA Purification System kit (Promega Corporation, WI, USA), as per the manufacturer’s instructions. Briefly, culture was pelleted and resuspended in cell resuspension solution by vortex and the cells lysed with lysis buffer. Alkaline phosphate buffer was added, mixed, and incubated for five minutes at room temperature. Neutralization solution was added, with the lysate centrifuged and the supernatant put into the column. The columns were then washed twice with wash buffer and the final plasmid DNA eluted with water. Plasmid DNA was diluted to 50ng/µL and sequenced using Sanger sequencing with the primers SV40pAnR for 3’ and pSelect for 5’ to confirm the TCR sequence.

Following confirmation of insert, 1µL of a 1:10 dilution of plasmid DNA was added to 100µL DH5alpha cells and heat shocked, as above. A single colony was added to 120mL of TB-zeo medium and cultured overnight at 37°C and 250rpm. Plasmid DNA was extracted using the PureYield Plasmid Maxiprep System kit (Promega Corporation), as per the manufacturer’s instructions. Briefly, cells were pelleted and resuspended in cell resuspension solution by vortex. Cells were lysed and incubated at room temperature before the addition of the neutralization solution. The lysate was centrifuged at room temperature, with the supernatant poured into the clearing column and vacuum applied. The column was washed with endotoxin removal wash, column wash, and then dried. Once dry, plasmid DNA was eluted with water.