If I understand you properly, you prepared a set of standard dilutions, then tested them (and worked, meaning by end-point PCR or by making a standard curve out of them?), then you stored them (in a freezer) and, when you tested them again, they did not work.

I had the same problem: I also prepared ready-made standard curves that I kept in the freezer. Only thing, the efficiency of the reaction based on the standard curve was always different. The problem is that the DNA degrades even if frozen, thus the low concentrations are likely to degrade and this skews the curve.

What they told me from the companies is to store the DNA at high concentrations and prepare the dilution afresh when required.

Hi,

here are two pitfalls to keep in mind

1. Concentration of plasmid standards is quite low. You should use buffers with carrier RNA for dilution series, e.g. Nucleic acid dilution buffer. This carrier prevents unspecific binding to the tube surfaces

2. Plasmids for standards should be linearized as secondary structures can affect the results quite significantly.

Best Sven

1) DNA at low concentration binds to tube walls. Use low bind tubes like Eppendorf LoBind. Use nucleic acid buffer - composed of carrier RNA (tRNA from yeast 30-100 ng/uL) diluted in TE (pH 8, TRIS 100 micromolar e EDTA 1 micromolar). Use the buffer to dilute your standard curve and stock plasmids. Be carefull with TE concentration!

2) DNA stored at high pH (8-9) and with EDTA is protected against acid hydrolysis and nuclease activity, yeast tRNA will maintain your plasmids free from adhering in tube walls.

3) Low concentration of plasmids in standard curve can give bad results (low reproducibility) because subsampling error and stochastic amplification/measurement. I Recommend the article: The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time.

4) Take extra care to not contaminate your samples and pipets with plasmids. Use filter tips and change your gloves after manipulating plasmids.

Best Jacó

Hi Mohammad, it is a very common problem when dealing with the storage of DNA samples or cDNA samples for somewhat long time, and often it is due to the low-grade material of storage tubes which can absorb and can even degrade some of the nucleic acid content over time, no matter the kind of freezing you are using for your samples. Try to use the following tubes, which may save your work;

https://www.thermofisher.com/order/catalog/product/AM12450?SID=srch-srp-AM12450

Also focus on the proper preparation methodology and pH adjustment of the buffer you intend to use for dilution and storage purposes.

Regards....