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weird problem with Primers! - problem with the gene sequence i used to design my primers (Jan/08/2008 )

Dear all,
i am currently having a very weird problem. I had designed 4 primers for a part of sequence of my gene of interest and then optimized all the primers, after which i got PCR products. After that i had to make a break and my work was transferred to someone else. During the break, the person sequenced the PCR product and found that it matched with my gene of interest. However, after i returned, the concerned person wanted to tell him what sequence i used to design the primers. I had marked and saved my primers on the sequence that i used. However, now when i am comparing the saved sequence with the sequence for the same gene in databases like NCBI and Ensembl, i am not seeing match!!!!!!!!! unsure.gif it is as if i designed primers for some other gene and not my gene of interest, which i m sure is not the case.
Please suggest me something!
thanks.

-sandesh-

QUOTE (sandesh @ Jan 8 2008, 08:12 AM)
Dear all,
i am currently having a very weird problem. I had designed 4 primers for a part of sequence of my gene of interest and then optimized all the primers, after which i got PCR products. After that i had to make a break and my work was transferred to someone else. During the break, the person sequenced the PCR product and found that it matched with my gene of interest. However, after i returned, the concerned person wanted to tell him what sequence i used to design the primers. I had marked and saved my primers on the sequence that i used. However, now when i am comparing the saved sequence with the sequence for the same gene in databases like NCBI and Ensembl, i am not seeing match!!!!!!!!! unsure.gif it is as if i designed primers for some other gene and not my gene of interest, which i m sure is not the case.
Please suggest me something!
thanks.


Is it possible that you are looking at sequence of some variant of the same gene (different species) and so the difference in sequence. Did you save the sequence of the gene when you designed the primers?

-scolix-

did you design your primers from outside the coding sequence?

-mdfenko-

QUOTE (scolix @ Jan 8 2008, 08:16 PM)
QUOTE (sandesh @ Jan 8 2008, 08:12 AM)
Dear all,
i am currently having a very weird problem. I had designed 4 primers for a part of sequence of my gene of interest and then optimized all the primers, after which i got PCR products. After that i had to make a break and my work was transferred to someone else. During the break, the person sequenced the PCR product and found that it matched with my gene of interest. However, after i returned, the concerned person wanted to tell him what sequence i used to design the primers. I had marked and saved my primers on the sequence that i used. However, now when i am comparing the saved sequence with the sequence for the same gene in databases like NCBI and Ensembl, i am not seeing match!!!!!!!!! unsure.gif it is as if i designed primers for some other gene and not my gene of interest, which i m sure is not the case.
Please suggest me something!
thanks.


Is it possible that you are looking at sequence of some variant of the same gene (different species) and so the difference in sequence. Did you save the sequence of the gene when you designed the primers?


Hi,
i had thought of the same possibility like u said, as the same gene exists in other species as well. The mistake that i did was - i compared my sequence with the sequence of the same gene in other species and therefore no match. Later on i repeated the blast with selecting the appropriate species. It gave 100 % match. rolleyes.gif thanks for your help to you both.

-sandesh-

QUOTE (mdfenko @ Jan 9 2008, 01:33 AM)
did you design your primers from outside the coding sequence?


I designed for coding as well as non-coding sequence but that was how it was supposed to be for my work. While performing the alignment i used the whole sequence (exons and introns) and not just coding sequence. So this problem can be ruled out. The problem is now solved.
thanks-

-sandesh-