Sequencing 40 kb long vibrio dna - Need some help on sequencing 40 kb long vibrio (Mar/17/2005 )

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

Dear Sequencer,

I think you gene fragment is simply too large. It might contain a lot of repetative sequence.

regards
yongyk

It seems to me that primer walking is rather inexpensive and fast when you start from multiple points in the fragment. I had to do this because my lab could not afford to sequence the large numbers of clones required to get significant overlap in a shotgun library. I am currently primer walking through a cosmid clone (~40 kb) but am starting in about 15 different places. Thus, each time I sequence from these positions I am getting about 5kb of new sequence and designing new primers. I used an AFLP method to get random starting points in the clone but the transposon insertion should work just as well. I should be done in about 8 or so rounds of sequencing and redesigning (primers are fairly cheap)