I would avoid any primer pair that yield hairpin loops, homo and heterodimers with a delta G below -5 kcal/mole (-6 and so on). Also, pay attention whether it generates dimers in which the 3' extremity can be extended, you'll want to avoid that.

I've been using this rule for a long time and it's been woking for me. In real time PCR, it's good to select primers that will generate an amplicon of 50-150 bp, with a TM ~ 60-65 C, and using the 3' region of the gene.

Hope it helps

I agree with Martin, specificity is the most important so as to avoid primer dimers or non-specific amplification.

Personally, I've always had pretty good luck using Primer3 (see attached link) for primer design. You can specifically Tm, size, or specific regions of your target GOI.

http://frodo.wi.mit.edu/

The info provided in this link will be helpful:

http://bitesizebio.com/articles/a-step-by-step-guide-to-designing-qpcr-primers/?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+BitesizeBio+%28Bitesize+Bio%29

This one is also good:

http://quantprime.mpimp-golm.mpg.de

If you want to detect gene transcript level, go PrimerBank http://pga.mgh.harvard.edu/primerbank/. Over 95% of the primers work well.

I apply the same rules as Matheus and that works for me.

Of course you need specificity, however, it does not sound like specificity is the issue here, it is the steps AFTER designing specific primers that is the issue. ie, are there any FURTHER criteria that should be followed.

Like Ryan has pointed out, Primer3 is a good primer design program to use, and I use the OligoAnalyser from IDTDNA.com to do the things you are trying to do.

I dont agree with the statement that "It's biology, not mathematics, numbers are mostly irrelevant." Its like a common undergrad saying that the students have chosen to do science, not maths, when you try and teach them the importance of applying statistics to their hypothesis testing. In this case the numbers are completely relevant, as they describe the thermodynamics of base pairing - the somewhat grey area is that it is not always clear how this exactly translate into either PCR success or failure. They should definitely be considered though.

Good luck in your primer design and keep us posted on how you go.

Cheers,

Hey Jasica,

Are you designing the primers for Sybr green based qPCR or Taqman based qPCR. In both cases, the specificity is very important. You have no need to worry about the secondary structure/dimer/self-dimer formation if you are designing the primer for Taqman qPCR but still you need to take care of secondary structure formation at the end of 3' position otherwise it will decrease your reaction efficiency. In case of Sybr green, you must take care of each and every secondary structure formation other wise the chances are very high that you will get the Ct value with even negative control after 30 or 35 cycles. There are many ways to increase the specificity and to optimize the reaction efficiency.

Let me know if you have any question.

Hi, upto my knowledge and opinion, the primers for RT-PCR should be designed in such a way that they would have following characterictics :

1.Their anealing site should be accross intron. (this will neglect the amplification of genomic DNA contaminantd if its present any how in your cDNA).

2.Amplicon size should be arround 100 - 150bp.

3.if you are doing RT-PCR for many samples, then try to design the RT primers of the same aneealing Tm.(1-2 degree diffrence is acceptable).So u can do all samples together.

These primers are comperatively very small than of GATEWAY primers. So there is almost negligble chances of formation of secondry structure and primer dimers. So no need to consider the rest of the parameters.

As already mentioned, specificity is the most important. If you want to reduce or avoid the formation of primer dimer you can include a 80 degree step after your elongation step!

Eileen Muhs how many time for this last step!?

Hi Jesica, you can just add the 80 degree step after your elongation. So for example your first cycle includes a 95 degree step and you carry out this step one time. Your next cycle includes your denaturation step (95), your annealing, your elongation (72) and your 80 degree step and you can repeat this whole cycle 35-40 times (all 4 steps) and at the end you will probably carry out a melting curve and after that a cooling step.

Hi hope this helps. If you have any further questions, than just ask.

@Eileen This tip looks really helpful...I'll also try in some reaction where I am facing problem of primer dimers.....

Is there any published report where this has been shown as advantageous to reduce primer dimer formations?

@ Ajay: primer dimers dissociate normally around 80 C if the primers are not GC rich, so we added in the Muyzer lab this 80 degree step. I just at a short look if there is any publication for this, but I haven't found any, however that does not mean that there is no one ;o) You can try to find a publication and if you can't find one, well than just try it and let me know ;o)

@ topic 80°C step: this only helps to avoid the detection of primer dimers. In case of very strong dimers or low amount of target you can still have a bad target amplification shifting your Ct values to higher values.

What are the limts for a good design regarding these 3 items? The program gives the Gibbs free energy for these three posibilities,There are a number beyond which it is not convenient to synthesize?

No one answered to these questions?