I want to design real time PCR primers for my gene of interest which is 1Kb in size and with one intron. What are the important parameters that I should keep in mind while designing primers? Is there any free software/database which is reliable in designing real time PCR primers?
http://blast.ncbi.nlm.nih.gov/Blast.cgi
www.ncbi.nlm.nih.gov/tools/primer-blast/
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
http://blast.ncbi.nlm.nih.gov/Blast.cgi
you can use the similarity between a set of genes (conserved region) of some organism, that similar to yours and which contain one intron. By the database in GenBank, pubmed you can design it.
http://blast.ncbi.nlm.nih.gov/Blast.cgi
www.ncbi.nlm.nih.gov/tools/primer-blast/
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Try to make your primers span the intron, this will help you keep residual genomic DNA contamination from affecting your results.
The size of your amplicon depends on the analysis method you will use. For real-time PCR analysis with probes, 150 bp is a suggested starting point, and the distance from the probe to the primer is important. Please research guidelines for the kind of probe you will use.
If you are using sybrgeen, then you should be extremely careful about the specificity of your primers. The 3' end of the primer (at least the final 3-5 bases) should not match to other targets in the genome (use the BLAST program suggested above to check this).You want as few incorrect binding targets for your primers as possible, and the 3' end is the most important.
You need to check for primer-primer and primer-probe interactions as well, so you should consider using a free online program like Primer3 to help you design the best primers. Primers used together in a primer pair should have a similar Tm and GC content.
The bioinformatic identification of sequences that may be good primers is only the first step, and I suggest you select at least 2-3 different primer pairs that meet the above conditions and then test them experimentally to determine which primer set performs the best.
Good luck!!
Becca
I agree with Virginia, she said it all. Primer3, or primer premier, or primer express software is user friendly. You just input sequence of your target gene in FASTA order. Software give you number of primer pair combinations and ranks them according to their penalty scores, The higher score the better primer. YOU must have in mind that in some cases even though it seems everything is fine, there is no 100% guaranty, so don't get frustrated.
IDT software is also great. If you use published sequences beware! Use BLAST as previously recommended.
Thank you ,for your answers,
I have designed some primers and I BLAST them with the Arabidopsis genome (I am working with Arabidopsis) and I am getting 4-5 non intended binding with other sequences. How to eliminate that? If it cannot please suggest me some tips to get a good q PCR results.
Thanks
Nikhil Job
The best way to design primer for real time pcr
Sigma oligoarchitect, SYBR, SCORPION PROBE, LIGHT CYCLER...
maaldrich.com/technical-documents/articles/biology/oligoarchitect-online.html
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