Hi there
I isolated bacteria from serum sample did the extraction of DNA for bacteria, followed by PCR. PCR band was of 100bp I sent it for sequencing. The results were approx 43 bp forward primer and 57 bp reverse primer. I reverse complemented the reverse primer seq and did the BLAST of both forward and reverse.
I got BLAST matches but for both there is no single match. The error message shown in BLAST search is, “No significant similarity found.”
I did following one by one;
1. I did the blast separately and also joined the two seq and did again
2. I did un check the low complexity region
3. I did the Mega-blast
but the result is same.
My primers were highly specific primers. I am suspecting following things;
1. Primer got attach to non-specific site for some reason and amplified junk.
2. During gel purification the sequence was denatured
3. It is actually some human intron sequence and not present on NCBI
4. And last but very least suspected it is some new sequence.
Help me I am lost and worried I have to submit my results so please help me.
Many Thanks