how to sequence 1500+ bp fragment? - (Aug/14/2007 )

I need to know the sequence of a fragment which is 1700 bp long, if i design (five) primers from 5'end, every 500 base, will that be ok to sequence the whole fragment??

Then i can combine all the sequences to get final, is it correct??

Any software which can do this joining??

Thanks for ur help.

I have never pcred across a fragment like that, but it should work. Have you thought about cloning the fragment into puc18 and end sequencing. The forward and reverse reads should give you about 1600 bp. Then you can design a primer, using the DNA from the original prep to finish the fragment. CAP3 is free and available at http://pbil.univ-lyon1.fr/cap3.php. (Huang, X. and Madan, A. (1999) CAP3: A DNA sequence assembly program. Genome Res., 9, 868-877.) and will work to assemble your sequences.

As jenvkuehl says, sequencing from both ends is a good approach, although you don't have to use pUC18 - just design forward and reverse sequencing primers at the 5' and 3' ends of your fragment. If you have the money to spend on it, many sequencing companies offer some sort of "gold" service, where they will start with a primer at the 5' end, obtain the sequence, then synthesise a primer to start sequencing again from the end of the first primer's sequencing and so on until the whole fragment in sequenced.

You could design primers every 500-600 bps (depends on how long the sequencing go and varies from sequencer to sequencer) and each of them for sequencing.

Wouldn't 4 primers work?

You could get Sequencher, free version and use it to analyze the sequences and compare the sequences. Its a good program.

if youhave clues on the sequence inside your vector, choose 4 primers, 2 per sense. Normally it should cover your sequence. A 5th primer may be used after.

In order o be sure, you may purchase 6 primers.

one you have the sequences, you can simply align two consecutive sequences and paste them in the correct position... at least, that's what I've done, though a specialized software might help