Hi guys......i am facing problems regarding developing standard curves for different copies of plasmid DNA using taqman probe real time PCR. i have successfully obtained standard curve for different dilutions of genomic DNA but when i used the different dilutions of plasmid dna i didnt get the results (the results for every dilution comes out N/A for plasmid dna).
i am using takara probe mix for this experiment, using FAM and TAMRA as flourophore and quencher dye.
the pcr machine used as CFX96 Real-Time PCR Detection System.
the pcr system i used 25ul
probe mix 12.5ul
probe primer 1ul (10uM)
primers 2ul each (10uM)
ddw 7.5ul
Shuttle PCR standard protocol
Sample volume: 25 μl
Step 1: 95℃ 30 sec
Step 2: PCR
GOTO: 39 (40 cycles)
95℃ 5 sec
60℃ 30 sec
If you have a right PCR product cloned into a plasmid, most of the time the assay will work with the same reaction and cycling conditions. If you didnt get any amplification for your dilutions I am suspecting something wrong with plasmid or the plasmid concentration. You can amplify the cloned fragment and sequenced to confim. Have you quantified the plasmid extration? You may need to repeate the plasmid extration if the concentration is too low.
Hello,
I have had good results with gDNA. Plasmid DNA could indeed be more difficult. Linearizing the plasmid should help.
Maybe this topic can help:
https://www.researchgate.net/post/Why_is_it_better_to_linearise_a_control_plasmid_before_PCR
and/or this paper:
Chen, J., Kadlubar, F. F., & Chen, J. Z. (2007). DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair. Nucleic acids research, 35(4), 1377-1388.
Best of luck,
I agree with Jeyaseelan and Simon. Simon is absolutely right that supercoiled topology of the plasmid template do interfere with efficient amplification. Although, this interference is more relevant for low copy number standards. We have also demonstrated this for conventional PCR (check the link below). For higher copy number standards, you need to get amplification, if everything is OK. Since you are not getting amplification with any of your standards, I suspect an issue with either the copy number calculation or the insert. I therefore suggest you to re-verify the copy number calculation and then carry out a conventional PCR (using the real time reagents) separately with insert-specific primers as well as vector specific universal primers. These experiments will help you to locate what is wrong with your procedure.
best wishes,
SD
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