Hi,
I am trying to design primers for attached sequence with resulting product length of 800-1000bp. But could not design any suitable primers. The tools I used are as under:
https://www.eurofinsgenomics.eu/en/ecom/tools/pcr-primer-design/#
https://bibiserv.cebitec.uni-bielefeld.de/genefisher2
Please suggest me any suitable link for primer design. Thnaks
I think the problem is likely that your sequences have very low GC content (14-15%) so the programs can't find any primers given the default parameters. You should be able to get something by lowering the minimum tm to something like 45°, raising the allowed maximum primer length, lowering the allowed GC content of the primer to something very low like 10-15% and lowering the melting temperature of the amplicon.
Personally, I use Primer Blast for most of my primer design off of genomic templates since you can tell it to check for specificity against a whole genome to prevent off target amplification, if you use the right settings.
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
It was able to find primers with ~50° tm for the Apis florea sequence by changing the Tm minimum to 45°, the optimum to 55° and setting the product size min to 700 and max to 1000. I don't know how good they are but it at least returns something to work with.
http://primer3.ut.ee/
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
Hello,
As Alexandra said your gene sequence has very low GC content. it will be difficult to design a primer or even if primer design is successful may be your PCR application is doubtful. In this case i would suggest you to use gene synthesis along with codon optimization service.
Hi, today I have tried to design with the first sequence from your .txt file. I've used http://bioinfo.ut.ee/primer3-0.4.0/ with subsequent analisys by http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form
Yes, oligos in not perfect but I have received a pair for 945 bp amplification and I am sure that it will be work after optimization.
Try it. If you have any questions, please write me.
One more comment regarding my attempt to design...you can add some LNA to eqilibrate the Tm of oligos. I usually add it in the first 10 bp of oligos. To estimate Tm and other features in detail you can use IDT oligoanalyser online tool
Thank you all for nice suggestions.
Dear Dr. Andrei BabenkaI, I also received same primer pair for 945bp amplification by using http://bioinfo.ut.ee/primer3-0.4.0/ .
How I will use the following website for subsequent analysis http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form
and how can I add some LNA (locked nucleic acid) to eqilibrate the Tm of oligos.
Will you please explain in detail?
thanks
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