Genomic Characterization of Metallo-beta-lactamases
Genomic DNA was normalised to 5ng/l with EB (10mM Tris-HCl). 0.5 l of Tagmentation Buffer (TB1) was mixed with 0.5 l Bead Linked Transposomes (BLT) (Illumina Catalogue No. 20018704) and 4 l PCR grade water in a master mix and 5ul added to a 96 well plate. 2 l of normalised DNA (10ng total) was pipette mixed with the 5 l of the tagmentation mix and heated to 55 C for 15 minutes in a PCR block. A PCR master mix was made up using 10 l KAPA 2G Fast Hot Start Ready Mix (Merck Catalogue No. KK5601) and 2 l PCR grade water per sample. 12 l of this mastermix was added to each well to be used in a 96-well plate. 1 l of 10M primer mix containing both P7 and P5 Illumina IDT UDI (Unique Dual Index) 10mer indexed primers were added to each well. Finally, the 7 l of Tagmentation mix was added and mixed. The PCR was run with 72C for 3 minutes, 95C for 1 minute, 14 cycles of 95C for 10s, 55C for 20s and 72C for 3 minutes. The libraries were quantified using the Promega QuantiFluor dsDNA System (Catalogue No. E2670) and run on a GloMax Discover Microplate Reader. Libraries were pooled following quantification in equal quantities. The final pool was double-SPRI size selected between 0.5 and 0.7X bead volumes using sample purification beads (Illumina DNA Prep, (M) Tagmentation (96 Samples, IPB), 20060059). The final pool was quantified on a Qubit 3.0 instrument and run on a D5000 ScreenTape (Agilent Catalogue No. 5067-5579) using the Agilent Tapestation 4200 to calculate the final library pool molarity.The pool was run at a final concentration of 750 pM on an Illumina Nextseq20000 instrument.
SUB15341736
PID-2012-E21_S19