Hi Nazek, what kind of sequence was supposed to amplify that primer? a protein-coding gene? a functional RNA encoding gene, like 16S or 18S?

I assume that you are getting the message "non significant result" from a tool like BLAST. Well, this depends on two things: first, your sequence, and second, the database you are using. And depending on the type of sequence, you have to select the appropriate database. For example, if the primer was suppose to amplify 16S or 18S, then an appropriate database would be Silva or greengenes.

Can you tell us a bit more about your sequence?

Hi , 

if you sequenced it just try to blast it in GeneBank which can match your sequence and identify it .See link below . Good luck 

Ali 

https://www.ncbi.nlm.nih.gov/guide/howto/find-published-info-gene-sequence/

Dear Prof. Nazek,

I understand your question. If it is only one gene you can repeat the amplification process again and try to sequence this product using either forward or reverse primer to check the quality of sequencing product.

second, did you find your primer sequence in the sequence that you get? if yes it is mean there are mutations in this gene and it will be very interesting. If not it means problem in sequencing.

Good luck and best wishes,

Hosny 

Dear all,

thank all for your collaboration and sorry for delay from my side, this is what we did;

1. we used primers from the article ; Diversity of CTX-M L-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. by Miche'le Saladin et al 2002.

2. we amplified a protein-coding gene, taille:543pb , the sequence is reliable

3. we used consensus primers (from this article) with high level of homology to the gene we looking for

4. no we did not find the primer sequence in our sequence, 

Yes the size matched the expected size

Yes it was direct sequencing of PCR products forward and reverse 

it was BLAST search

I agree with Prof Artur. In addition I suggest you that when doing a Blastn against nr database, search your sequence individually with all the three programs, i.e. Highly similar sequences (megablast); Optimize for More dissimilar sequences (discontiguous megablast) and Optimize for Somewhat similar sequences (blastn). You can find these options in the Program selction section just above the BLAST button. While doing a Blastp, search with all the options i.e.  blastp (protein-protein BLAST), PSI-BLAST (Position-Specific Iterated BLAST), PHI-BLAST (Pattern Hit Initiated BLAST), DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST)

Thease are also placed just above the BLAST button. In addition, try to search homologous sequence through ORFfinder or cdd search.

https://www.ncbi.nlm.nih.gov/orffinder/

https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

I hope you will find smething interesting. using these tools we recently characterized a novel Insect specific virus, which initially gave "no matches" a lot many times. Ultimately identified thrugh less stringent (somewhat similar sequences) search.

If you feel comfortable, you can send the sequence.

Best wishes,

SD

the obtained sequence is an amplification of a beta-lactamase encoding gene (blaCTX) but we didn't amplify the entire coding sequence of the blaCTX-M gene.

For the detection of this gene,consensus primers were chosen from region with high levels of sequence homology to the blaCTX-M gene.After sequencing the PCR products obtained we used simple blastn program to look for sequence homomlogy with the other blaCTX-M gene.

there isn't an intact ORF present in the obtained sequence.

We will see what we will get with the recomended programs: 

 (megablast);  (discontiguous megablast) and(blastn)