Optimum Parameters (Primer Dimer Dg, Harpain Dg, Tm And Something Like This) For Primer Designing For Real Time Pcr (Syber Green I)

Question

[Mohammad Reza Bakhtiarizadeh]

hi all I want to design primers for my genes by Vector NTI software for real time pcr (syber green I). i know that there are some papers and texts about that but there are different information about the parameters. if possible let me know about optimum parameters for designing primers by this software. i want to know your experiences about it.

regards

[Michael]

well it depends ... on your data, and why don't you have time to read some papers? also why do you tag this with primer3 if you want to a proprietary software? third why do you want to use this software, you are possibly not getting a lot of feedback here, because not many will have access to it.

[Mohammad Reza Bakhtiarizadeh]

I said that i want to use other members experiences about that and also i read the papers and texts. however i think that a real experience can be more useful than theoretical information.

[Michael]

then I would recommend rephrase your question not specific to a certain software but specific to RT-PCR. Also, as I said, it depends on many factors, example, which temperature range is required by the machine what kind of organism, etc.. I thought you could at least give some more specification.

[Mohammad Reza Bakhtiarizadeh]

I am confused because for example Tm temperature for a spacial primer is different in various software and i think that vector NTI has a more similar algorithm to real situation. my tissue is fat and liver (sheep). my main problem is what parameters are good to primer designing.

[Michael]

come on, there must be somebody knowing more about rtPCR primer design?

Answer 1

[Michael]

Sorry, I am really not an expert on this, but if nobody else can help, I just tell what I would do int this situation...

I think this is a good and very concise first read:

http://emboss.open-bio.org/wiki/Appdoc:Eprimer32

Actually, it is worth noticing that, most important parameters are not free-to-choose parameters but constraints. These constraints are defined by your real-time RT-PCR protocol. Say Tm, primer length, primer GC.

• Read your protocol or machine manual or ask the person running that machine, or call the vendor, that will tell you most constraints, so the optimum constraints are those which are optimal for your protocol. Then use these optimum constraints as parameters. Say, the optimal Tm is 58 degr, set set Tm range to 57.5-58.5, size must be 20, set the size range to 20-20, if the optimal product length is 250, then.... Use the region which you want to amplify, maybe spanning an intron to be able to distinguish DNA contamination.

• Then use primer3 web to iteratively try out these parameters. Leave all the parameters you don't know about as default. If you don't find any primer, relax the constraints a bit. The problem might be in general that with the optimal parameters you don't get a primer!

• This is very important: After getting a primer pair, run a sequence search eg. using BLAT against the genome if available, and vector and contaminant databases. If the primers are not unique, try again.

I hope this helps.

[Mohammad Reza Bakhtiarizadeh]

thanks so much for your guide. regards