PCR amplicon, pooled competition assay. Replicate2, Time point 4
Genomic DNA (gDNA) was isolated using the Blood & Cell Culture DNA Mini (<5e6 cells) Kits (Qiagen, cat. no. 13323) as per the manufacturers instructions. The gDNA concentrations were quantified by Nanodrop. For PCR amplification, 1 g of gDNA was amplified in a 200 l reaction using Q5 High-Fidelity 2X Master Mix (NEB #M0491). PCR master mix (100 l Q5, and 10 l of Forward universal primer, and 10 l of a uniquely barcoded P7 primer (both stock at 10 M concentration). PCR cycling conditions: an initial 30 sec at 98 C; followed by 10 sec at 98 C, 30 sec at 68 C, 20 sec at 72 C, for 22 cycles; and a final 2 min extension at 72 C.