Question

Hi, I'm new here. Currently, I'm doing transposon mutagenesis of bacteria. I have tried direct sequencing of transposon flanking region,but it didn't work. I'm trying arbitrary PCR now. However, I'm facing problems of getting bands. Can someone please help me out by giving some suggestions regarding sequencing of the transposon flanking region or arbitrary PCR method? Thank you

Answer 1

@rko86 Is splinkerette-pcr method like Inverse PCR which involves digestion and ligation before amplification?

Answer 2

Hi Sulli_lim, When you say that you tried direct sequencing and it didn't work, could you be more specific about exactly what you tried? It would help people answer if you either list your protocol (brief), or give a reference paper that used the same protocol. I have had good results getting the location of Tn5 insertions in genomic DNA in the past. It took several tries to get results. I harvested the genomic DNA from mutants of interest, then digested the DNA, then cloned the digest fragments into a standard cloning vector. One important step was doing size fractionation of the digested DNA. Without this step, I had no sucessful clones. Once you have clones with the appropriate resistance markers (one from your transposon, and a different resistance from your cloning vector), you can sequence directly from your cloned plasmid DNA.

Answer 3

Hi JeanD. Thank you for replying me. I used the sequencing primers provided in the kit and directly sequence the transposon flanking region without undergo any PCR step. Firstly, isolate the genomic DNA, then sequence it. The primers will bind to the mosaic ends of transposon. I'm refering this paper: Transposome insertional mutagenesis and direct sequencing of microbial genomes.