3 replies to this topic

[fzhang]

Hi, everyone. I had some abnormal dissociation curves when performing my qPCR today. I drawed a schematic figure of my curve because I could not get a snapshot of the original curve presented on the ABI 7000 SDS system.

Why there is a such a high half-peak in the initial part of the curve? Is this because there is too much template in my tube?

Thanks a lot in advance.

Attached Files

•  curve.bmp   576.05K   142 downloads

Edited by fzhang, 30 May 2010 - 12:58 AM.

[lsek]

Hi,

You could try "Print Screen" or PrtScrn using keyboard, and then open MS Paint. Right click a mouse and choose "Paste". You will get the picture on the computer screen. You can then crop the pic or just save it in JPG or BMP.

From the look of the sketch, it could be unspecific amplicon at lower Tm of the dissociation curve. Normally, I do dissociation curve at temp 55 degree Celsius to 95 degree Celsius. Can't comment much without knowing the lower Tm of the range you used for dissociation curve.

Ohya, you can run agarose gel to look at the product. That will tell you what is wrong.

Edited by lsek, 30 May 2010 - 09:28 AM.

[fzhang]

lsek, on May 31 2010, 01:25 AM, said:

Hi,

You could try "Print Screen" or PrtScrn using keyboard, and then open MS Paint. Right click a mouse and choose "Paste". You will get the picture on the computer screen. You can then crop the pic or just save it in JPG or BMP.

From the look of the sketch, it could be unspecific amplicon at lower Tm of the dissociation curve. Normally, I do dissociation curve at temp 55 degree Celsius to 95 degree Celsius. Can't comment much without knowing the lower Tm of the range you used for dissociation curve.

Ohya, you can run agarose gel to look at the product. That will tell you what is wrong.

thanks. I have attached the original picture. and my dissociation is from 60 celsius to 95 celsius. Thanks a lot.

Attached Files

•  curve.bmp   1.92MB   137 downloads

[lsek]

Not sure what how to explain from this figure. You only have two templates? Did you include non-template control, as well? How did you derive the DNA template (e.g. 1st strand cDNA synthesis, genomic DNA, etc). If you still have the qPCR product, you can try running on agarose gel and have a clearer picture.

Also, you can try running another primer set that have been shown to work before (or template). That will ensure that the reagent is still working, or the machine is not faulty etc.

Good luck.