I inherited a set of primers for rat COX2 and thought I'd see if it worked on mouse samples. I used Primer BLAST to put in the sequences, specified mouse, and got back results indicating that my primers would not work. Just for fun, I repeated the BLAST for rat, and the results don't indicate that COX2 is a target. Am I using the right program to look for primer templates and amplicons?
Hi,
If you know your primer pair is going to amplify a particular DNA segment (COX gene of Mouse in this case), then I would just retrieve the DNA of interest (coding sequence, free of introns I guess?) and check the primers have enough homology to anneal and amplify the target DNA. If it doesn't match, I would say it is not specific for mouse gene and I would design a new primer. You really can do it manually!
Thank you all! I had to laugh when I read Ananda's, "You really can do it manually!" My reaction- "Manually?? What is this 'manually' that you speak of?" You're right, of course, and I should have done that first.
Merin and Artur, I inherited them from a student who recently graduated from my lab, and I've trusted her work but my mentor says, "Trust but verify!"; I should be more careful about the 'verify' part.
Your advice is greatly appreciated!
Have you added the restriction sites to your primers, probably due to presence of these additional nucleotides you are not getting the results.
Manually- when you have AAAAAAAAAAAAAAAAAAAAAAs in your primers, that has to match TTTTTTTTTTTTTTTTs :) I guess you dont need a program to verify this ;)
Hi, Dr Anne Tye .
i ask you this primer for specific DNA , cloning (cDNA) or for real time PCR?
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