PCR was run with SYBR Green Taq and three different template DNAs that are wild type, heterogeneous, and homolozygous for a SNP. The only difference between the tubes is the template DNA.
The homozygous tube has a fluorescence starting around 40 at cycle zero. What does this suggest about the amplification? Why is the fluorescence so high initially?
Any advice or ideas would be much appreciated. Thank you!
Edit: I repeated the amplification and got the same result. The DNA is from the Coriell Cell Repositories and is reported to have good 260/280 and 260/20 ratios. Perhaps there is another SNP in that DNA that is inhibiting the primer annealing on that template?
Since the only variable in the preparations is your DNA template, it is possible that the DNA from that sample has a contaminant with a high background signal. I am not sure how you extracted your DNA, but if the DNA contains a substantial amount of remnant phenol, chloroform, or ethanol... these could potentially lead to a high background signal. Additionally, something could have just gotten into that specific well (dust, cotton, etc.) of the plate that has a fluorescent signal when hit with a laser. Last, it could be a simple pipetting mistake where you added a significantly higher amount of DNA in that well compared to the others to start.
I think your first step is to simply repeat the experiment on a different plate, and in different wells (in case the detector in that well is the culprit). If you see the exact same thing, you can drill down to find out what is happening in the DNA sample. Run it through a nano-drop or spec to see the UV fluorescence and make sure the 260/280 and 260/230 ratios are good. If not, you may want to run your sample through a DNA clean-up column to rid any potential contaminants. Hopefully that solves your problem!
Good luck,
~Adam
Hi Molly,
I also agree with the suggestions above by Adam....Your three DNA preparations should be very similar in quantity, purity and integrity. ....It will be a good idea to repat the experiment again. The issues related to quanity, quality and purity can be easily assessed by checking q-PCR of any other locus in the three samples which is exactly same in sequence...In that case your amplification plots should be nearly identical...If yes, then you may think of possibilites (as mentioned by you) other than just DNA quality realted issues.
In addition you can also look forward to design a specific probe (instead of non-specific SYBR gree) in the region of interest . This will also help to get rid of non specific signal...
bye
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