4 replies to this topic

[GordonLeung]

Dear all,

I am trying to amplify my target gene from viral DNA for making a standard curve. I have used qPCR (Sybr system) instead of ordinary PCR to monitor the specificity. Regarding the dissocation curve, the peak corresponds to genomic DNA template has a melting temperature at ~81.5 oC.

After that, I have purified the PCR products using gel purification kit. A 10-fold serial dilution of the amplicon have been done and they are subjected to a second qPCR (with the same reagent and thermo-cycling profile as the previous one). Surprisingly, everything goes well execpt the peak at the dissociation curve has shifted to ~85 oC. Is it contamination? Or the melting temperature of my target gene has changed after purification? Attached please see the dissociation curve for that. (I'm sorry for the low resolution)

I have identified the amplicon by RFLP and it is the gene of my target.

Comments are always welcomed!

Attached Files

•  dissociation curve.bmp   380.63K   87 downloads

[Trof]

Did your run both products on gel/compared size?
Tm can sometimes shift in different conditions, but 4 degrees is AFAIK a bit much. However diluted PCR aplicon is surely a different condition.

[GordonLeung]

Thanks Trof

I've tried comparing their sizes and seems that they are more or less the same length.

I totally agree with your comments. I've repeat it three times and same result is coming out.

[Trof]

Originaly there are guidelines for preparation of DNA standard curve templates. Usually the amplicon is cloned to plasmid and maintainedin this shape for long term storage. Plasmid is then cut with a single cutting enzyme and concentration is measured after purification. With known legth of the plasmid you can calculate copy number. Then you dilute your cut plasmid with buffer containing "dummy" nucleic acid (rRNA) to compensate for the low complexity of template.

Maybe such preparation including dummy rRNA would give similar Tm to your samples.

[GordonLeung]

Thanks for your information and it's helpful and inspirative