I am working on detection of rickettsia in ticks, while I use a primer whose annealing temperature in the original paper 48, I always get non specific bands without appearance of the specific one
If I increase the annealing to 55 or 56 will this increase can get me the specific band ? for your information I dont have a positive control
You should run a gradient PCR over a range of temperatures to determine the optimal Tm for your primer set with your specific starting material. I usually run a gradient from 50-70 over a range of about 10-12 samples to determine which temperature works best for me. A higher Tm will typically be more specific, but may also amplify nothing if it is too high for that primer set. Also, if the primer amplifies non-specific bands across the entire range tested you may have to develop a new, more specific, primer pair.
Dear Ronel
I tried to increase anneling fr 48 to 54 by two degree each time however, this were not affect the non specific bands ( nearly 4 in each sple I tested) only in 54 one non specific band appears and others vanshied
While temperature 58 led to disappearance of all the non specific bands at all but primer dimer still existed in the sample
My question is : does anneling at 58 in this situation mean that primer work?
I use half if the tick after longtudinal dissection
The parasitima is high in some ticks and low in other according to the puplished papers but I always measure dna in extracted sample via nanodrop before use in PCR
Positive control is diffecult to supply by companies and I always run PCR with negative one
I will suggest to apply a gradient PCR Reaction and set the temperature from 54 to 62 oC and then see which temperature is the optimum for your primer annealing. I hope you will get your desired band by doing so.
Set up a gradient PCR (choosing a gradient from about 50 to 60 ) to determine the optimum annealing temperature for the primers. You should be able to get an optimum that eliminates non-specific bands
- Badges
- Science method