I agree with Primer3 plus software being great, but strongly recommend you to use NCBI's improved version called 'Primer BLAST'. The key advantage is that it will BLAST your primers against the reference genome, so you make sure that they are specific to the amplicon you want to get.
If by any chance you wanted to read some background info on primer design, I published a short article on the subject where I explain the basics.
Primer3. I don't change any parameters. Just paste in my DNA sequence, select amplicon size and go. If it's for RTPCR and I want primers in different exons, it's easy to mark on the sequence the position of the exon intron boundary to request primers on different exons.
In case of qPCR primer pairs (for TaqMan or SybrGreen) I use the tool at the website of Integrated DNA Technologies. It works quite well.
Primer3 its online available and easy. Preset parameters are good enough.
I keep getting an error message when I click on pick primers after i have pasted the sequence: Error = Unrecognized base in input sequence
You shoud use FASTA format for input and if you have used FASTA format then check if you have any space or character in the sequence
Hi, an overview: http://molbiol-tools.ca/PCR.htm
I also use Primer3.
Cheers, Nadine
Hi I prefer this one it is so easy. just you need to choose the sequence and copy it
http://depts.washington.edu/bakerpg/primertemp/primermelttemp.html
and to check the complementary
http://arep.med.harvard.edu/labgc/adnan/projects/Utilities/revcomp.html
Good luck
Good morning.
When I was designing my primers, I found that CLC Main WorkBench was a perfect tool to design primers and assess primer parameters, due to its easiness of handling and a good visual interface for users. The bad part is that it isn't a free-software, so you'll have it in a free mode for 1 month, and after that, you can use the basic tools (not include Primer Design), but if you can buy a license, it would be perfect!
We use SE-Central. Although you mostly download your sequence in that and you can design your own primers. You can also do some cool in-silico analysis like plasmid construction, restriction enzyme digest analysis, molecular cloning etc.
I agree with Primer3 plus software being great, but strongly recommend you to use NCBI's improved version called 'Primer BLAST'. The key advantage is that it will BLAST your primers against the reference genome, so you make sure that they are specific to the amplicon you want to get.
If by any chance you wanted to read some background info on primer design, I published a short article on the subject where I explain the basics.
I very much agree with Ruben Alvarez-Fernandez' comment. I have designed many primers with that strategy. It is necessary to check how many products a primer pair can amplify. If more than one (with adequate sizes), then it shouldn't be used.
I strongly recommend the Vector NTI software, from Invitrogen
Hi
I recommend the OLIGO5 or 7 software. They present all the essential choices. Of course it is necessary to check the primers (designed using any software) in the target genome using Primer-BLAST which provide by NCBI. This step helps to avoid false priming.
If you like, I can send the software to you.
God speed
I've used Primer Premier 6 for a long time. It's easy and convenient to operate.
Thank to you all. Bahar Zare, you can send me the software as well. Thanks again.
I found very useful this paper, where you can find all those tricks to design good primers
http://www.ncbi.nlm.nih.gov/pubmed/24011032
I also recommend Primer Blast because you can use it to check if undesired products from possible contamination could cross-react with your desired target, in case of cultures.
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