qPCR standard curves - how can I keep the quality good? (Jun/28/2005 )
Dear all,
Recently I am frustrated by the quality of my standard curves that I prepared for my qPCR experiments.
At my lab all the standanrd curves are stored at 4 Celsius, made with genomic DNA or cDNA.
For my experiment my standard curve is made of PCR product. And I also stored it at 4 Celsius. ( Because I am afraid the freeze-thraw staps may break down my the PCR product.) Last Thursday I test the curves with qPCR and I saw a perfect parallel amplification plot and linearity.
Today I perform my experiment with it, I saw half of dilution serie has shifted to a higher CT values! Does this mean I need to prepare fresh std.curves for every experiment? But it is not practise, it takes me half hour to do that, and I need to test the linearity before running the qPCR with it [and I have to do many qPCR everyday 
] May I store it in freezer? How many time may I freeze-thraw my curves?
Any inputs will help me a lot, thankyou
Recently I am frustrated by the quality of my standard curves that I prepared for my qPCR experiments.
At my lab all the standanrd curves are stored at 4 Celsius, made with genomic DNA or cDNA.
For my experiment my standard curve is made of PCR product. And I also stored it at 4 Celsius. ( Because I am afraid the freeze-thraw staps may break down my the PCR product.) Last Thursday I test the curves with qPCR and I saw a perfect parallel amplification plot and linearity.
Today I perform my experiment with it, I saw half of dilution serie has shifted to a higher CT values! Does this mean I need to prepare fresh std.curves for every experiment? But it is not practise, it takes me half hour to do that, and I need to test the linearity before running the qPCR with it [and I have to do many qPCR everyday
] May I store it in freezer? How many time may I freeze-thraw my curves? Any inputs will help me a lot, thankyou
What buffer are you diluting your standards in? For a 4 degree storage recommend that you use TE otherwise can see some loss especially with the very dilute samples. What I ended up doing is making a very large batch of my standard curve and then aliquot into small volume (0.2 or 0.5 ml tubes) enough to do two - three points with allowance for pipeting loss and freeze aliquots. Then when do run, just defrost one set of standards and discard any remaining. This way avoid repeated defrost/freeze cycles
Dear Ribosoul,
What i did was, i prepared a standard stock with concentration up to 5e10, aliquot into several tubes and store in -20oC.
I will take out one tube prior to use, defrost and perform a 10 dilution and only use 5e6 to 5e1 as standard.
Later, the remainer of standard stock 5e10 can be stored in +4oC for a week.
In order to get a perellel amplificaion on standard curve, you have to perform serial dilution freshly.
I am looking forward for input to improve storage condition of standard.
Any suggestion will be greatly appreciated 
Best regards
Hadrian
Hey, i am wondering how you can quantitate your diluted samples to know their specific concentration? Should you do the spectrophotometer measuring or do you calculate their concentration based on the dilution factor and your original stock sample?
THe problem is the stability of PCR products, the extremities 5' and 3' have tendancy to be degraded with time. To pass over this problem you should add some nucleotides, usually 10, at the 5' and 3' extremities of your PCR product. For this, order primers with these nucleotides and then synthetize your amplicon.
I performed this many times and stability of PCR fragments is very good, I keep them at 4°C. They are as stable as they had been cloned in plasmids.
Recently I am frustrated by the quality of my standard curves that I prepared for my qPCR experiments.
At my lab all the standanrd curves are stored at 4 Celsius, made with genomic DNA or cDNA.
For my experiment my standard curve is made of PCR product. And I also stored it at 4 Celsius. ( Because I am afraid the freeze-thraw staps may break down my the PCR product.) Last Thursday I test the curves with qPCR and I saw a perfect parallel amplification plot and linearity.
Today I perform my experiment with it, I saw half of dilution serie has shifted to a higher CT values! Does this mean I need to prepare fresh std.curves for every experiment? But it is not practise, it takes me half hour to do that, and I need to test the linearity before running the qPCR with it [and I have to do many qPCR everyday
] May I store it in freezer? How many time may I freeze-thraw my curves? Any inputs will help me a lot, thankyou
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Dear Season,
Yes, as you said I measure the concentration of the oroginal stock and culculate the standrads concentration base on their dilution factor. To quantitate every diluted samples is not wise to carry out.
Dear Tomy,
Thank for you input, you suggestion to add 10bp at both 5' and 3' extremities had greatly improved my standard stability.
Thank you 6.022x10e23
Best regards
Hadrian