Experience Help needed with Taqman primer/probe design

[emucaki]

These are all questions that you usually don't find in a rulebook, and I believe are questions which answers come from experience.

I am currently designing probes and primers for the first time using PrimerExpress. I am aware of all the rules, such as as small of an amplicon as possible, probe 10ºC higher than primers, etc.

My problem is that a region I want the probe to cover is GC poor and I can only get the probe Tm at 65ºC. I am aware of MGB probes, but lets just ignore those for now. I have to have primers at a Tm of 55ºC which is kind of low. I could have the primers at 56 or 57, but would that have a huge negative impact on my results?

Also, would the results vary for a primer pair in which the forward primer is 10nt from the 5' end of the probe, while for another pair, the forward primer is 15, 20, or even 30 away from the probe (but the amplicon is the same size).

On a related subject, say I wanted to do a multiplex PCR and two of the primers overlap, causing competition. This would cause problems for the reaction, right?

Is there any reason why the Tm's calculated by Primer Express is off compared to ones calculated by online tools such as OligoCalc? OligoCalc/Oligo 1.0 tend to show a 55ºC as 52ºC and my 65ºC probe a 60ºC.

It also shows a 25bp primer with 44% GC has the same Tm as a 24bp primer with 38% GC content, how does that make sense?

[danfive]

I have to have primers at a Tm of 55ºC which is kind of low. I could have the primers at 56 or 57, but would that have a huge negative impact on my results?
A: Pick the best primers and probe (don't design around a perfect 55 C Ta), any Ta in that approx range is fine.

Also, would the results vary for a primer pair in which the forward primer is 10nt from the 5' end of the probe, while for another pair, the forward primer is 15, 20, or even 30 away from the probe (but the amplicon is the same size).
A: Very easy again, don't worry about this, it is not a problem (they don't overlap).

On a related subject, say I wanted to do a multiplex PCR and two of the primers overlap, causing competition. This would cause problems for the reaction, right?
A: Yes, if your primers overlap you cannot proceed with multiplex PCR.

Is there any reason why the Tm's calculated by Primer Express is off compared to ones calculated by online tools such as OligoCalc? OligoCalc/Oligo 1.0 tend to show a 55ºC as 52ºC and my 65ºC probe a 60ºC.
A: Different logarithms (calculations) that are proprietary, so each group designs its own. Ask the nearest pro around you for what they use and use it as well.

It also shows a 25bp primer with 44% GC has the same Tm as a 24bp primer with 38% GC content, how does that make sense?[/QUOTE]
A: Could be weak software or the sequence of the GCs can make all the difference, for example if the GCs are clustered together that makes them stronger structurally than separate Gs and Cs.