Hi there, try with a software Serial cloner. I think your primer's length is significant enough to self-annealing. Try to reduce a small stretch of primer design.
Dear Mohamed,
I think the program is not fixed. some factors can alter it, like used Enzyme and its stability. I recommend you to read this article by Thermofisher, hope it will help you.
https://www.thermofisher.com/jp/ja/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/rt-education/reverse-transcription-setup.html
It's Tm is 66.9°C, so a couple degrees less then that should be your annealing temperature. To be absolutely sure you should perform a "temperature gradient" on a couple of samples since it will also slightly depend on your PCR kit and your thermocycler. However, 31bp can potentially be too long for a primer - it can anneal to itself. Ideally, try to design specific primers between the size of 18-22bp.
Dear Mohamed
Reverse transcription enzymes usually work in 37-42 °C and common reverse transcription kits have a high temperature (about 70 °C) to annealing in the first step and then chilling on ice to base pairing.
I think you can easily follow your enzymes protocol.
Dear Mohamed,
1. The sequence is able to form a hairpin or a dimer with a helix of four basepairs. One can easily check for such things by a dotplot program, like http://www.biophys.uni-duesseldorf.de/html/local/DOTPLOT/dotplot.html
2. A denaturation temperature (or annealing) temperature, calculated either as a mean value for the full sequence or as dissociation/association temperature might be misleading, if the 3' nucleotide(s) of a primer denature at lower temperatures, as is here the case for the 3'A. One can easily check for such things by a program that predicts the cooperative denaturation behaviour of double-stranded nucleic acids, like http://www.biophys.uni-duesseldorf.de/html/local/POLAND/poland.html
3. Your primer is indeed quite long and might tend to partially bind to other sequences. This risk can be reduced by increasing the temperature (if possible) but increases the problem mentioned in 2.
Probably, the pairing temperature is close to or equal to Tm, which is 66.7 ºC. But you must consider the Tm of the other primer.
I think your primer sequence is too long, forming a primer dimer
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