Sequencing Analysis - (May/27/2006 )

Hi!

I'm having trouble analyzing the sequence of my pcr product.............since this is the first time I'm trying it. A few basic doubts to clarify.

My pcr product is 159bp. After forward sequencing, I tried BLAST but the only compatible result with the gene of interest was my antisense primer (22bp) in the sequence.

My question is, is the result acceptable?
Does the primer sequence (sense & antisense) retains in the pcr product?

Desperately needs help!

Thanks.

What was the quality of your sequence? Did you BLAST it as DNA or as translated DNA? Did your initial PCR, from which you presumably gel-purified the product for sequencing, show multiple bands?

As to the other part of your question, yes -- the reverse primer's sequence will be detectable in the forward sequencing run, and the forward primer's sequence will be detectable in the reverse sequence run -- the primers are incorporated into the PCR product...

Hi,

Finally someone to share my problems on sequencing. Im doin biodiversity studies, so i do need to sequence out as many possible bacteria's to create a phylogenetic tree.

My fragment size is 973bp. I can get good readings in my"anti-sense" primer it can read up to 700bp. But my supervisor wants me to sequence both sides. I did n the results for forward is not clear only bout 200bp iscan be obtain. This reallly slows down my work.

I did post my enquiry online n got help wher i was asked to down load the assemblyLIGN but i cant seem to get a free version of the soft ware.

My question is as same as yours whether is it enough to sequence per direction. Will question arise when im writting up my thesis? Is it crediable this way?

Pls help!!!!!!


Thanxs

Did you try a different forward primer? Often repositioning the primer (even by a basepair or two either way) will overcome such problems. Can you post your primer sequences here?

Thanks.

The sequence was good with minimal noise around 140 bp. The BLAST was in nucleotide-nucleotide search engine. The pcr product was clean with single band at 159 bp.

Everything's okay. Just that after BLASTing the only compatible sequence was 22 bp, which is the antisence complemented sequence.

Btw, my sense primer: 19 bp
antisense primer: 22 bp

Help needed.

I've tried blastx as well..........same results. Anyway, I'll try to sequence it again. Probably, this time it will be okay. Thanks for the input.

Cheers.

I can recommend you very good software to do reverse complement for a sequence:
DNA Baser
You can use it free.
It is easy to use it and it has a very nice interface (Mac style).



http://www.dnabaser.com/intro.swf