Does anyone have the experience of detecting the activity of DNA polymerase besides radiolabelled method? I have tried Picogreen but the results could not be stable. Does anybody help make sense of this?
Hi there,
Measuring pyrophosphate generated by polymerization, there are some commercial kits for this.
Here are some possible reasons for instability of the PicoGreen signal.
1. The DNA is becoming degraded due to the presence of DNase activity in your samples. Your DNA polymerase may be contaminated.
2. The PicoGreen is sticking to the sides of the cuvette or assay plate. If your are using plastic cuvettes or assay plates, reduce this problem by including 0.005% Triton X-100, Tween-20, or Brij-35 detergent in the assay buffer.
3. The PicoGreen is becoming photobleached due to excessive illumination. Minimize the exposure of the dye to light, including room light.
There are other non-radioactive methods you could try, such as the one I invented, which is also fluorescence-based:
http://www.ncbi.nlm.nih.gov/pubmed/16266678
To Adam B Shapiro:
Thank you very much for your kind reply. Can you send me the full text of your paper,please? my e-mail : [email protected] looking forward for your reply.
Best regards!
I suggest that you read the work.
PLoS One. 2017 Sep 1;12(9):e0184162. doi: 10.1371/journal.pone.0184162. eCollection 2017.
Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization. Olszewski M, Śpibida M, Bilek M, Krawczyk B.
Wang he Hello ! I am trying to find out Unit of my DNA POLYMERASE too. Do you by any chance have full text of the paper you mentioned , Fluorescence-based DNA polymerase assay by Tveit and Kristensen.
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