Hi everyone
I tried to do PCR, and I expected to get a band in 121, but is my third PCR that I have gotten 400bp band. I Checked my primer and all of the other parameters. it should be mentioned that only one time I got the band in the right place with this primer.
but after that I saw band in 400bp.
Does anybody can help me?
Thanks In advance
Roxana Nezami
What are your primer sequences?
What is your organism?
What is the source (tissue, colony, DNA extract, cDNA) of your template?
You can use a service like NCBI Primer Blast to test for possible alternative products with your primers. See the following page and the link thereon.
https://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/
Hi Roxana
solutions could be numerous, lack of specificity in your primers, contaminations of your samples, protocol inducing muletiers amplification, structural variation at site of your amplicon...
as David said, we need more details. sequences of your primers and species are requested in order to help.
all the best
fred
How did you come up with an expected size of 121 bp?
Could be as simple as the difference between cDNA & gDNA as a template.
Also, double-check your band size in your ladder. Many look very similar, even though they have different size bands.
David F. Barker Thanks David
My sample was the eggs of Ancylostoma caninum which has been isolated from feces.
And my sequence is related to ITS2, is a region that help us to identify the parasite species.
Frederic Lepretre Hi Frederic
As I explained for David I tried to do a PCR for ITS2 region. and this primer is specific for this region.
And today I tried another primer which is related to other speices (uncinaria stenocephala) and I got this bands.
the expected size is 524 bp.
can I conclude that I have gotten band from this gel or not?
Roxana Nezami
Have you tried to optimize your primers by running them at different annealing temperatures? You might be able to do some in silico checking for PCR specificity at: https://www.ncbi.nlm.nih.gov/assembly/GCA_003336725.1/#/st
On this page, at the right, click on the "Run Primer-Blast" link. At the destination, enter your primer sequences in the boxes near the top and click the "GET PRIMERS" button near the bottom of the screen. This could give you an idea of what alternate products might be obtained with your primers in A. caninum. I found the attached paper interesting too--it specifically mentions the use of gradient PCR to optimize the amplification.
Katie A Burnette I have gotten this size from NCBI.
Thank you for your answer and recommendation.
Dear Roxana Nezami since you are working with eggs isolated from dog feces, and the primer is targeting the ITS region. This region sometimes vary between the different parasite species of the same genus. I had the same experience when working in Echinococcous spp. eggs. So as recommended earlier you can do sequencing. Also, consider the other species that could be present in the dog feces when performing the primers BLAST. You might see an expected product size of 400 and 524 bp related to specific species.
- Badges
- Science method