I have been using Clone jet PCR cloning kit with a pJET1.2 blunt as a vector. The insert is from a clean PCR product that went through a blunting reaction (supplied with the cloning kit). The E. Coli used for the process is Subcloning Efficiency DH5α Competent Cells. In this protocol, colonies are only formed by bacteria transformed by an insert carrying plasmid, but in any case, I have proven the insert is there through conventional PCR. The miniprep product is used as a positive control in Real time assays, as the insert contains in it the sequence I am looking for. For at least three different systems, I get relatively high miniprep yields (3000-5000 ng/ul), but when I run the calibration curve, the 10^3 ng/ul sample gives me a Ct value around 30. At first I thought it is an issue with the primers I designed, but running one of the samples (150 ng/ul) from my experiments got me a Ct value of around 23, plus, as I said, this happened in three different systems. I can't figure out why this would happen. Any suggestions will be welcome.
Thank you
Ori Elad
Try to scale down template as you are most probably inhibiting reaction. When I used plasmid as template I used 100 pg as highest amount.
"3000-5000 ng/uL" is very high, even for a midi prep. RNA contamination is probably throwing off your quantification - I'm assuming quantification was performed with spectroscopy (e.g. Nanodrop)? Try quantifying using fluorometry (e.g. Qubit, picogreen) using a dye that is specific for dsDNA. Just adding RNAase to the plasmid prep won't fix quantification using UV-Vis. Nucleotides absorb very well.
Eliminate RNA contamination and quantification errors as your problems.
Good luck.
I agree with Alberto and Nick. 3000 ng/ul is extremely high template concentration and inhibiting for PCR or qPCR reactions. first try to eliminate RNA by RNaseA treatment and chromosomal DNA contaminations in your minipreps by running your preps on agarose gel, extract the supercoiled ds pDNA fraction of your plasmid containing insert from the agarose gel and purify it. Then measure the A260, A280, followed by copy number calculation using the following online tool.
https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/dna-copy-number-calculator.html
in this application, set the calculations for custom DNA fragment, enter the total length of your plasmid (vector+insert) at the Fragment length space. Enter your prep concentration (after RNase & chromosomal DNA elimination) and you will get the approximate copy number of plasmid (in other words your copy number of your insert) in your original prep. Based upon this results make appropriate dilutions of your plasmid DNA prep in suitable buffer to get a dilution series containing 10^12 to 10^0 copies/mL. from this series use the 10^7 to 10^0 copies/ml as template for your qPCR as calibrators. Please run each dilution in triplicate.
I would be happy if you get success in your experiments. Please let me know.
Best wishes,
SD
Ribonucleotides left over from RNAse digestion will absorb at A260, so either purify the prep further (column, re-precipitate) or use a specific quantification method for DNA - fluorometry is much more accurate and sensitive than spec measurements.
I would check your miniprep on an agarose gel. I suspect that you will find that a large part of your material is not plasmid.
Nonetheless, you will probably also find that you are overloading your PCR. It's a good idea to convert your DNA concentrations to absolute numbers of target DNA molecules. There used to be a formula for this in the Applied Biosystems Catalogue.
Using more than about a million copies in a real time PCR is excessive. Be aware that it the Ct value is too low, it may interfere with the background subtractions (which usually uses the first 15 cycles for background subtraction. I recall a clinical sample that was unusually full of Bordetella pertussis. It had Ct=9 and the curve looked seriously abnormal until we corrected the background subtraction.
Also, if you're getting incomprehensible real time PCR results, running a 10x dilution series of your material is a good idea. The amplification curves should be evenly spaced (ideally differing by Ct = 3.3) and parallel on a logarithmic plot.
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