Posted 17 March 2005 - 12:02 AM
Hello! I need someone to help me out with sequencing my 40 kb long vibrio dna. i did the shotgun technique, sonication of my dna to 1 kb fragments, digestion with SmaI, digestion with a variety of enzymes... among others. Now, my professor suggested that i digest the dna again with several primers, have the dna isolated and inserted to a vector then analyzed. thus creating a group of smaller clone fragments from the previous one. my problem is, i have been encountering the same problem since day 1 of my sequencing, the sequences tend to be on the same site, not representative of the parent dna. i am now using the ez-tn transposon technique from epicentre to sequence the smaller fragments of dns, however they still tend to result to sequences of the same site... i have been doing this since last year and i really wanted it to at least work out... i am considering the use of genome walking but i guess it would be way too expensive... HELP OUT GUYS! Thank you!
Sequencer from Osaka University