When I perform qPCR using SYBR green, I get amplification in every plasmid DNA. I have used plasmid DNA as a template (1 microlitre) and also water (1 microlitre) instead of template (control). Because it was doubtful I extracted plasmid DNA and also water and all those were providing signals (Ct values). I am using Pb18srRNA as a primer. I also try with nuclease-free water in control wells, but still I am getting the Ct values (around 27-29) for water. When I run the samples on 2% Agarose gel, I also got band in case of water. I have attached here the results.
Well Well Type Threshold (dR) Ct (dR) Details
A8 Unknown 1857.202 27.43 Water
B8 Unknown 1857.202 27.01 Water
C8 Unknown 1857.202 10.33 Plasmid DNA
D8 Unknown 1857.202 10.01 Plasmid DNA
Can anyone please help me to get rid from this??
Talha
Do you have a picture of your agarose gel? The Ct values look very fine for me, there is a difference of ~17 between DNA and control, thats very good. I think the bands of the control reactions you are seeing on the gel are your primers ("primer clouds"). What size have your control bands? What is your amplicon size?
Right, so either you see primers (i got those too at the bottom of 1,5% agrarose), primer dimers or look for really fine double-destilated-and-all-that-stuff water, like water for injection. Also, you didnt mention that so i guess it's worth to remind - use laminar air flow safety cabinets :-)
Make sure, tips should not be contaminated
Use laminar air flow safety cabinets or careful with breathing while pipetting....
- Badges
- Science method