GSM5729344: Input_2; Porphyromonas gingivalis; Tn-Seq
Genomic DNA was also isolated from the bacteria recovered from abscesses as well as from the input P. gingivalis library. DNA was shearded targeting 200-600 bp sing a Covaris sonicator. The sheared DNA was purified using a Qiagen PCR cleanup kit to remove te buffer. The DNA was used for C-tailing with Tdt and a mixture of dCTP and ddCTP optimized to give about 18 C residues. This reaction was cleaned up using a column and the product was used for PCR with a primer that binds inside the transposon near to the insertion site and a second poly G primer. The resulting PCR products (200-700 bp) contained the amplified DNA at the transposon insertion site and were then gel purified. Library was constructed using a Nextera XT Kit (FC-121-1012).