quad tgRNA prevention (rpsl replicate 1)
50 ng of purified E. coli genomic DNA was subjected to 10 cycles of PCR using the respective pair of target-specific primers. After purification with a 1.5X ratio of Ampure XP beads (Agencourt #A63880), 4-8 additional rounds of PCR were performed to add the appropriate Illumina indices for multiplexed sequencing. A final cleanup using a 1.0X ratio of Ampure XP beads was performed prior to qPCR quantification and pooling. In all cases, Phusion DNA Polymerase (NEB #M0530L) was used for the first round of PCR, the KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems #KK4602) was used for the second round (indexing) PCR, and the KAPA SYBR FAST Illumina quantification kit (KAPA Biosystems #KK4824) was used for library quantification. Sequencing was performed on Illumina MiSeq instruments with the v2 chemistry.
quad-rpsl-rpsl-1