Go to General se ngs: a) Product size range: Size of your amplicon. OpWmal amplicon would be ~ 120 bp. b) Amplicon of 70-â200 bp are acceptable. You could ...
Primer design using Primer 3 Plus
Rat Genome database: Gives rat data including genomics, gene:cs, physiology and func:onal data. Also, has informa:on on other species: Mouse, Humans and Chinchilla Go to www.rgd.mcw.edu
Click on Gene search
Choose Rat from the species drop down box
We entered GAPDH. It returned 253 GAPDH genes found in rats.
When you click on your gene of interest it shows all the informa:on associated with it.
Scrolling down you will find Nucleo:de Sequence linked to the gene
Clicking on the nucleo:de sequence opens up the sequence on NCBI Genbank.
Copy the FASTA sequence and either save it as a text file (recommended) or copy to clipboard
Open hTp://www.bioinforma:cs.nl/cgi-‐bin/ primer3plus/primer3plus.cgi/
Paste your sequence in the “source sequence below”. Or you can also upload the saved text file. You could also give it a sequence ID (some name)
Go to General seZngs: a) Product size range: Size of your amplicon. Op:mal amplicon would be ~ 120 bp. b) Amplicon of 70-‐200 bp are acceptable. You could also give different product size range seperated by a comma. Eg: 70-‐200, 80-‐150. This will look for primers first from 70-‐200 bp and then look for primers between 80-‐150.
c) Primer size: Op:mal length of primers is acceptable as 18-‐28 bp in length. d) Primer Tm: Min = 57, Max = 63 and Opt = 60. Maximum Tm difference = 3. This is the difference between the forward and reverse primers. e) Primer GC%: Min = 40, Max = 60 and Opt = 50. The GC content of the primer is sued to predict the annealing temperature. Op:mal GC% of primers are between 50-‐60%
Go to ADVANCED SETTINGS: a) Number to return: The number of primers would be returned aher search. Set to 10 (less or more depends on the user). b) Max 3’ Self complementarity: Set to 0. If no primers are returned increase it by 1 un:l you get primers. The 3’ end of the primer shouldn’t be complimentary to each other or it would result in primer-‐dimer forma:on c) Max Poly X: Runs of 3 or more Cs or Gs at the 3’ end should be avoided as their presence may promote mispriming at C or C-‐rich sequence. Set the value to 1 and increase by increment of 1 if there are no results
Aher making the seZngs, click on Pick Primers and it would display the primers
Check on all the primers you are interested in tes:ng and click on Send to Prime3 Manager
It would open a new tab with the selected primers.
You can click on CHECK on each primer and check for any errors
Addi:onally, you can BLAST all the primers and check
Aher running BLAST, look for your accession number and check for the E-‐value and Query coverage. Generally, the Query coverage should be 100%
References and Further reading • Real-‐Time PCR (qPCR) Primer Design Using Free Online Sohware: Brenda Thornton and Chhandak Basu • qPCR guide: Eurogentec • qPCR technical guide: Sigma-‐Aldrich
