primer efficiency

[ashu330327]

Dear Sir
How to calculate primer efficiency by qpcr using SYBR GREEN I?

Thanks

Reagards
Ashu

[mchlbrmn]

You run a standard curve, in duplicate or triplicate, of a series of dilutions of a positive template. For example 10^-1, 10^-10, 10^-100, 10^-1000, (10^-10000). The PCR machine software can usually give you the efficiency number, or give you the slope from which you can easily calculate it. This efficiency number is good for unkown samples who's values fall with those values seen in this standard curve. You should also verify that the curve is fairly even, not abnormally low for 1/2 and abnormally high in the other 1/2 of the curve, giving a perfect 2.0 efficiency overall (or 1.0 depending on how they define it).

[ashu330327]

Dear Sir
I am in trouble while not getting proper amlification curve. My question is that not getting good amplification curve in target unknown sample but got gud curve in target post callibrator sample.

Target post callibrator sample and target unknown sample are same sample but they have different gene. In callibrator sample hOUSEKEEPING GENE IS USED AND IN TARGET SAMPLE CRTE1 gene is used.in target sample zig-zag pattern is obtained but not in the case of callibrator sample.
What could be the cause behind this reaction. I have diluted 5 times cDNA (5ug RNA is used to make cDNA). 1ul(cDNA) was taken in 10ul reaction

But when I optimized the primer of CRTE1 gene in general PCR machine for specific annealing temp. I got good amplified product after 30 cycles.

Cycle condition is

94--------------30''
62--------------30''
72---------------30''

size of amplified product is 130bp.

cycle condition for qPCR

95-----------------10''
62------------------10''
72------------------12''
FOR 45 CYCLES

I dont known what is happening . Shall I change the cycling condition of qPCR? or something else is disturbing the reaction
Please tell me sir.!!
Any advice would be highly appreciated...

[mchlbrmn]

Hello. I have PCR experience, but I'm not really an expert. I don't quite know what you mean by, "post callibrator", but I think I understand that your problem is that the housekeeping primer set works, but the target gene primers do not work in qPCR, but they do work in conventional general PCR. If you want to optimize the qPCR this is best done in the same qPCR machine with the same reagents as will be used in the final experiment, unless you can not easily use the machine. If you cannot optimize in the qPCR machine, then it is best to use the qPCR reagents in the conventional machine. The PCR conditions can be quite different with different reagents. I use the Roche Lightcycler 480 system, and when I use their reagent the melting points are higher than with a more common taq polymerase mix by several degrees, due to higher salt I believe. So, if you were using my system, the problem might be fixed by raising the annealing temperature in the qPCR.
I don't understand what the problem is with your qPCR. I don't understand what you mean by a "zig zag" problem with the amplification. Did you get a good single melt peak? Did you run the qPCR sample out on a gel? So, I don't quite know the problem, but I do know that it's not surprising that the results were different in conventional PCR, as the conditions can be quit different with a different enzyme mix.