GSM8338235: plasmid DNA sequencing, UTRome, plate A; Escherichia coli; OTHER
Plasmid DNA was extracted from E. coli with the Promega PureYield Plasmid Midiprep Kit. To determine the appropriate ratio of DNA to Tn5 transposase, 50 ng of each plasmid pool was combined with various dilutions of in house purified Tn5 transposase in a 25 µl reaction (reaction conditions 10 mM Tris pH 7.5, 5mM MgCl2, 10% DMF) and incubated at 55°C for 8 minutes. The tagmentation reactions were stopped with 2.5 µl 1% SDS. The optimization reactions were PCR amplified and ran on a 6% PAGE gel. The reaction that yielded the most library in the size range 400 to 1000 bp was selected for full scale amplification. Five µl of the tagmentation reaction was PCR amplified in a 50 µl reaction with Phusion polymerase for 7 cycles, ethanol precipitated and size selected (400 to 1000 bp) on a 6% native PAGE gel.