I'm working with genotyping of SNP's of some drug metabolism-related genes. I've already ran 2 sucessful genotyping assays before, and now, this third SNP that I'm working is making me worry about it. I added some photos of the plots.
In the pdf, there are images from the genotyping assay, divided by a blank page because it was made in different days. In the first part, there is a plot of a failed sample that not amplified, and in the next page, another one that amplified. Same order in the following part. What could be an explanation for this? I'm using the same samples of my first two good assays (those that ran great). The kit that I'm using is one commercialized by Applied Tech. for this specific SNP assay. From a recommendation, I've already raised the denaturation time to the following conditions below. Should I raise the elongation time to 1:30min too?
My current PCR conditions are: 60*C 2min (UDG activation), 95*C 15min (Initial denaturation and activation of the Pol.), 50 cycles of 95*C 45 secs and 60*C 1min, finally, 60*C 1min (Final elongation). 12ul of reaction (10ul of Mix and 2ul of DNA).
Please, help!
Hello,
I think that there is a serious problem with amplification or probe system. Honestly your charts does not look like the one which works well because ROX dye fluorescence is too high (and you use NFQ so you actually can use low ROX mix). Could you check the product with gel to know which the problem is. If you see the amplified product on the predicted length on both chart, please check the quality of the probe that you have.
hope that it helps.
Really, really, Thanks! I got more idea of what's happening now. I was kind of surprised when I saw that results because I've used the same assay (I mean, the same tube containing the probes) like 3 months ago with other samples, and regarding as you mentioned, the ROX dye fluorescence was normal in that assay that I did 3 months ago.
The fact that the ROX dye fuorescence was too high is related to the quality of the probe? that means that my probe is already hydrolised? How are that two phenomenas related (just for curious)?
I will run the gel as you told. Is it possible to run Taqman probes in agarose gels? It will be the first time that I will try.
Note: I'm adding the results of the same assay with different samples, look that the ROX dye fluorescence is lower than the alleles. So this means that there is a possible trouble with the probes, right?
The intensities of the fluorescent probe on Taqman assay is normally measured as the relative intensity of ROX fluorescent. ROX is added in the reaction mix, as a reference dye to normalize the different fluorescence between each well on the qPCR.
I think that the probe fluorescence is constant, so it is possible that the amplification is not going well, or the probe does not carry dye any more. Please check the amplification goes well by checking the PCR products. Check if the probe is already hydrolyzed or not, it is a good idea that you check the probe quality by gel, but I think you'd better do it on PAGE.
good luck!
Try calling Life technologies and ask them for the expected amplicon size (if you don't have the primer design file for the assay), then run the reaction product on a gel to look for the amplified product. I've had to do this previously, and the product size was 60bp. It turned out that the probe wasn't binding properly, and Life remade the assay and it worked fine. The fact that the assay worked fine only 3 months ago and now doesn't work is a bit of a worry. Maybe just re-order it?
try KASP™ genotyping assays that are based on competitive allele-specific PCR and enable bi-allelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci. www.lgcgenomics.com
this chemistry hasSuperb accuracy and performance
Accuracy: >99.8% based on independent assessment
Industry leading SNP & InDel assay conversion rate (>90%)
Thanks, guys. Thanks, Michelle. Because of your message, now, I remember that there is a CD that comes with the products when you buy it, and I guess that there is the information about the primer and the desired amplicon. I'm going to check the content, and then, I'll run the gel and verify if it's something with the probes.
Thanks, Atilla. My current lab was working with that kind of SNP assays since 5 or 6 years, I guess,, and we never considered the choice of changing. So, as you mentioned, I'll search a little more about the specifications of KASP™ assays and its benefits, because we're designing a new project in SNP's, so this could be the chance to try it out. Thanks for you suggestion, I'll be talking with my lab coordinator.
I really appreciate your help, guys!
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