I need to design primer collagen 1 for Real time pcr which would work for mouse, rat and human. how I will design any suggetions?
If you are new to molecular biology and dont know how to design primers, better to use already published primers. There are several primers for your target which have been published for the rat model. And They should be working for the human host as well. You can easily check it on primer-blast.
Most (not all) of mouse and human can be found on this site Primerbank.webloc. They are good.
Also you can design thru NCBI site which is linked to primer software by default by following these steps:
*In google, type NCBI
*Select "Nucleotide" option
*Type name of "gene" and enter; option of "species" shows up
*Select "species" several sequences shows up
*Open one of sequence by clicking the top row
On your right hand side of screen there is is option, "Pick Primers"
*Click "Pick primers"
*Enter any parameter you want otherwise Click: "get primers"
you can copy the sequences get it made from idt or some company.
Generally I make 2 sets and test them, and keep the best, Most of them they work good
That is correct, but also in NCBI- Pick primers is linked to Primer 3 software. Means you do not have to cut and paste sequences, you just click Pick primers, sequences are transferred automatically from NCBI to Primer 3 software,
Cheers
Hi!
Just the basic:
1) First take the sequence you need from database (NCBI...) in fasta format. Take the same orthologous gene from rat, mouse and human.
2) Align them (nucleotide sequence) using MUSCLE ( https://www.ebi.ac.uk/Tools/msa/muscle/ )
3) Take the conserved region/regions and design primers. You can use IDT primer quest online software (https://www.idtdna.com/pages ). Test for primer dimers and haipins with oligoanalyzer tool (IDT).
4) Blast your primers in https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome to be sure about specificity.
Other important details: The primers are in the same exon or in different exons? you want to amplify just one gene isoform? Genes are really the "same" (orthologous)?
Best
Jacó