I have T-DNA insertion mutant line of Salk_057742 (shows T-DNA is inserted in Exon) line. I have done DNA genotyping and it show perfectly fine mutant, as i am getting band in Lb+RP (no band in LP+RP). I have made cDNA from RNA and checking for transcript amplification by using gene specific primers (made from exon exon junction) i am getting CNGC13 gene amplified in this mutant. I am not understanding how it could be possible and how to troubleshoot whether its a mutant or not?
Thank you
I wonder if those are primer-dimers. Why not cut out the bands, clean up, and send for sanger sequencing to know what they are?
Hi Ramgopal,
as mark said be sure of the size of your amplicon and use the good ladder to new that. The second point is that maybe your sample is not homozygote for the mutation and you're amplifying the wild type genotype. You must therefore have 2 bands. waiting a result from a non amplified fragment is dangerous, you should also use an internal control PCR to run as multiplex PCR for your study (the amplicon length must therefore be distinct to your target amplicon) as in this protocol (Article Amplification-Refractory Mutation System (ARMS) Analysis of ...
)fred
.
Hi Mark,
my amplicon size is around 150 bp and it is coming on respective position in that gel. I have also tried to amplify full length gene as well and it is getting amplified properly in mutant.
When i am trying to perform experiment on this mutant i am getting significant difference in phenotype.
Hi Frederic,
when i have done DNA genotyping i have used 12 plants and all were showing homozygous mutation. In this ladder i have shown only two samples but i have done this genotyping in 8 samples and all are showing gene specific band. i have done another pcr where internal control and my gene bands are of different size and i am again getting the same results.
I repeated it so many time and every time i am getting the same results. But i will also try with positive and negative control.
Hello Ramgopal,
where are your primer for your RNA located? Before, or after the insertion? If your t-DNA is inserted into an exon, there might still be a partial transcript, which might, or might not be functional.
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