Hi,
I am an ecologist researching bats - I collected some feces of bats and I want to make sure they are coming from the species I am looking for. Inorder to do so I want to amplify a "barcode" region in the Mitochondria from the DNA in the feces. I am trying to choose the right primers for this job from two options I have:
Leray forward primer GGWACWGGWTGAACWGTWTAYCCYCC
Leray reverse primer TANACYTCNGGRTGNCCRAARAAYCA
16S forward primer CGGTTGGGGTGACCTCGGA
16S reverse primer GCTGTTATCCCTAGGGTAACT
First I checked if they match the species I am looking for (Pipistrellus kuhlii) and if the sequence they amplify is specific to this species, sure enough I got a 100% match and the primers matched well.
Here comes my problem, I wanted to make sure they would also amplify other species of bats incase I collected the wrong feces. So I searched for all 33 bat species that appear in my study area and downloaded their COI sequence and 16S sequence in order to see if the primers match on them.
I am using Geneious to check this:
I entered a sequence of the Forward primer and also the reverse primer (and choose "R.C - reverse complement selected sequence"). The primers didn't match perfectly on most species - with differences in b.p from 0-10 which is quite a lot I assume - since the primer sequence is quite short by itself. I also had quite a few times when I aligned the primers on a bat gene that the reverse primer matched before the forward primer - should that create a problem? I mean wont they be going the wrong way?
Hope my question is clear and someone can help me
Thanks in advance!
I am an ecologist researching bats - I collected some feces of bats and I want to make sure they are coming from the species I am looking for. Inorder to do so I want to amplify a "barcode" region in the Mitochondria from the DNA in the feces. I am trying to choose the right primers for this job from two options I have:
Leray forward primer GGWACWGGWTGAACWGTWTAYCCYCC
Leray reverse primer TANACYTCNGGRTGNCCRAARAAYCA
16S forward primer CGGTTGGGGTGACCTCGGA
16S reverse primer GCTGTTATCCCTAGGGTAACT
First I checked if they match the species I am looking for (Pipistrellus kuhlii) and if the sequence they amplify is specific to this species, sure enough I got a 100% match and the primers matched well.
Here comes my problem, I wanted to make sure they would also amplify other species of bats incase I collected the wrong feces. So I searched for all 33 bat species that appear in my study area and downloaded their COI sequence and 16S sequence in order to see if the primers match on them.
I am using Geneious to check this:
I entered a sequence of the Forward primer and also the reverse primer (and choose "R.C - reverse complement selected sequence"). The primers didn't match perfectly on most species - with differences in b.p from 0-10 which is quite a lot I assume - since the primer sequence is quite short by itself. I also had quite a few times when I aligned the primers on a bat gene that the reverse primer matched before the forward primer - should that create a problem? I mean wont they be going the wrong way?
Hope my question is clear and someone can help me
Thanks in advance!
