I want to design primers for my cDNA. What I should to do?

Question

I want primers that exactly amplify the target.

Answer 1

Do you know what the target is? If you do then you can design primers off of something related on GenBank, then try them on your sample. If you don't know what the target is, then you have to build a cDNA library, then sequence it using plasmid primers.

Answer 2

If you can use pubmed then go and find the mRNA sequnce for your gene, cut it out including the \\ at the end, and paste it into primer3. To find this just google primer3 and click on the first link, its an MIT site. Anyways paste the sequence in and tell the website how big you want your product to be, you can specify exactly what regions need to be included or excluded and where the primers must start and even end too and the melting temp of the primers. Once you get the primers, it will be easy since this is the gold standard primer design software and is very user friendly, do a quick blast search to make sure they do not have high homology, over 18nt, to another gene. Primer3 will give you multiple primer pairs with variable GC content. The best is to design your melting temp at 58-60C and the GC between 40-60% and have low secondary structure and no primer dimer. Also do hot-start PCR and have an DNApol that has 3-5' exonuclease activity, like a HiFi/Platinum Taq or KOD Hot Start Polymerase. Cheers.

Answer 3

you should go to www.idtdna.com
they have primer designing programs.

but if you want to design your primers exactly, then all you have to do is take 17-22 nucleotides from the ends of your cDNA. For the first primer you can just read it off the sequence, but make sure for the second primer, you do the reverse complement!

the main problem in designing primers this way is that you are selecting specific sequences that may differ vastly in their temperature of melting (Tm) and thus your PCR may not work because the temperature you set for annealing may not be applicable for both primers. Therefore you may want to consider adding additional bases to the 5' end of each of the primers to even out the Tm. Remember these bases will be incorporated into your final PCR product. If you are doing cloning using restriction enzymes, you could add restriction site sequences to the ends of your primers to get a fragment that is easily clonable into a vector. Good luck!

Answer 4

Assuming you have the sequence and this is just standard PCR (not RT), simply take the first 15-20 nucleotides of the sequence as the 5' primer and then using the last 15-20 nucleotides, determine what the reverse compliment would be and this will be your 3' primer.

Reverse compliment:

For the sequence - 5' AGATTGAC - 3' , the reverse compliment would be GTCAATCT.

Next, run both primers through some web-based analysis such as http://seq.yeastgenome.org/cgi-bin/web-primer and evaluate the G/C content, annealing temperature, etc.

For more info or if you run into problems, you might try reading:
http://www.mcb.uct.ac.za/pcroptim.htm

Good luck!