You must linearize your plasmids or the differences in plasmid coiling will affect your results.  Also, if your plasmids have gone through multiple freeze-thaw cycles they will start to degrade.  You should really aliquot them into small batches that you thaw once, use, and toss any remaining.

In agreement with Katie above regarding the need for linearization - you would also want to linearize always using a specific restriction endonuclease that cuts at a single site so you have control over exactly where each plasmid is cut. Other means of linearization that you are proposing would be too sporadic in the resulting plasmid fragments and breakage points; and could even end up breaking inside the insert/polycloning region as well.

If I understand correctly, your qPCReactions containing ~100,000 plasmid copies (assuming a single target insert in each plasmid) generated Cq values from 31 to 35. Normally Cq values from 31 to 35 would indicate copies of ~128 to ~8, respectively.  100,000 copies should appear about ~21.4 Cq given 100% reaction efficiency, and a 1-copy estimated intercept of about 38 Cq. 

If indeed your reactions generated this result, it could be that the plasmid prep carried out by the other laboratory (without inspection by gel electrophoresis) is diluted in a buffer that is still inhibitory to your qPCR.  Or, quantification is off - you could be overestimating the starting material concentration. 

Supercoiled plasmids (as you know) can stall the qPCR up to 7 cycles because of lack of primer access to target sequence.  Even if so, you would expect Cq values of about 28 instead of 31-35 at 100% efficiency, but you could see these higher Cq values if your reactions were only 70% efficient for instance.

The high SD among triplicates may be because thermal breakage of plasmid constructs is a random/stochastic process that will differ from well to well - even given the same reactants.  If you prepared your triplicates in a singe tube (all qPCR components and plasmid present) and then pipetted into the 3 wells on the plate from that single tube, you should theoretically see better triplicate agreement.  However, if you pipetted into the triplicate wells as separate entities, this would invite larger errors/Cq disagreements for certain.

Dear all, 

Thank you for the insightful responses. 

Dear Jack M Gallup,

May I know how the values in "If I understand correctly, your qPCReactions containing ~100,000 plasmid copies (assuming a single target insert in each plasmid) generated Cq values from 31 to 35. Normally Cq values from 31 to 35 would indicate copies of ~128 to ~8, respectively.  100,000 copies should appear about ~21.4 Cq given 100% reaction efficiency, and a 1-copy estimated intercept of about 38 Cq." were derived?  

From past experience, 100000 copies gave Ct values of 18 to 22, varying with the plasmids and different primers. I suppose that is due to pipetting errors during dilutions, differences in PCR efficiencies, or quantification. Quantification was achieved using fluorometry (Qubit or Quantifluor).

Working through the basic log based math involved in estimating initial copies in a qPCR reaction using standard curve based interpolations and/or extrapolations, one invariably arrives at the simple expression:  Cq = -logEamp(copies) + b  which easily rearranges to Xo = EAMP(b-Cq)

You won't see these exact equations published anywhere but in a few things I've published including a 2011 book chapter.  However, these relationships are suggested in different forms throughout the qPCR literature - just not as succinct.

In your example above (wherein you state 100000 initial reaction copies appearing anywhere from 18 to 22 Cq), those (if 100% efficient reactions [e.g., EAMP = 2]) would indicate reactions wherein the b (y-intercept values) were 34.609 and and 38.609, respectively. 

However, it is unlikely that one would ever get a y-intercept as low as 34.609 (see publications by Jo Vandesompele on this: e.g., the lowest possible Cq value for one copy at 100% efficiency is thought to be 35, however, I have always found one copy reactions to report nearer 37, 38 or 39 given 100% reaction efficiencies).  So the more reasonable value for your 100000 copies at 100% efficiency would be the reaction series wherein you generated the y-intercept value of 38.609; putting your 100000 copy Cq value squarely at 22.

In the equation above:

Xo = initial reaction copies

EAMP = exponential amplification value = 10(-1/m)

b = y-intercept of standard curve of: log of copies vs Cq

Cq = the arithmetic average of observed technical replicate Cq values for a sample

Simple substitution into this equation and/or rearrangements thereof will allow you to look at whatever feature of qPCR math you wish to in a straight-forward manner.

Xo = EAMP(b-Cq) generates the exact same result(s) as the Livak/ABI User Bulletin #2 inferred relationship:  Relative quantity = 10((Cq-b)/m)

When b = the Cq value for 1 copy (y-intercept), then both equations solve for initial reaction copies, but when b = the y intercept of a relative dilution standard curve of Cq vs the log of the dilution factor, both equations generate identical unit-less relative quantity values that can be compared with one another.

If you work with these relationships enough, it becomes more readily apparent what the implications are.

Finally, realize that a range of 18 to 22 Cq is not an acceptable range of difference for a technical replicate of some kind for calculating/estimating a reliable copy number.... that represents at least a 1600% difference from one end to the other... very dangerous degree of variation probably only explained by faulty quantification of the standards, since one would surely physically notice a pipetting error that large.  Proof of the homogenaeity of the linearized plasmid standard is very important in your studies, as would be accurate/precise quantification of those plasmid standards before deployment into qPCR.  These are what the tea leaves read for me here on this anyway.

Finally, if you have indeed achieved a y-intercept of 34.609 on some occasion, that would represent one of the best, most sensitive qPCReactions I've ever heard of.  Depending on how low you can reasonably draw the quantification threshold line also determines this value (and all threshold line-based Cq values for that matter); so that is a factor as well...

Dear Nathanael

Several times, I am using plasmids both as qPCR controls and as calibrants.  Some plasmid solutions are Certified Reference Materials at 2E+06 (from Joint Research Center, IRMM, Belgium) and they are of very high quality allowing for PCR efficiencies ranging from 95-105% and R2>0.98. Recently, I prepared a plasmid control with an insert of 1100 bp and still I can get the same performance values (Eff. of 92% and R2>0.99). My plasmid solutions are stabilized with boiled salmon sperm DNA and stored at -70 ºC. You can also stabilize them with other non-recombinant, different plasmids having a different origin of replication. STorage at -20 ºc is also possible but there is some little degradation. At 4 ºC we observed a clear degradation. During the first three months, Ct values increased approximately 1-1,5 cycles. I never linearised the plasmids.

Hoping this will help you.

Regards

Excellent information Eugénia,

Perhaps some supercoiled plasmids are more prone to stalling the qPCR than others then (see attached).  Which seems entirely reasonable; as each situation could most likely be unique.  In Nathan's case, there are still some unknowns that, perhaps when solved, will shed more light.