real time qPCR standard range plasmid - (Feb/15/2015 )

Hello,
I'm training in a virology laboratory and I am working on adenovirus . I'm trying to quantify the increase of Adenoviruses genome in various samples by real-time qPCR . To do this, I use a standard range made ​​from a pEX -A2 plasmid with an insertion of the gene of interest.
This plasmid was provided to me by an industry that synthesized it in vitro.
After dilution of the powder provided in an ultra-pure water, I linearized and quantified the plasmid using a NanoDrop (I obtained 4.6 ng/µL) , the NanoDrop detection limit was 2 ng/µL.
Then, to produce my standard range, I calculated the number concentration copies/µL, I got about 7 x 10 ^ 12 gc/µL. Then I diluted for a standard range of 1 x 10^7 to 1 gc/µL.

I performed a qPCR to test the standard range and I could detect samples up to 10 gc/µL.

Then I froze my samples at -80 ° C.
The following PCR , I was unable to detect 10 gc/µL .
Then the following PCR , the standard range came out with very different Ct of the first PCR: I was around 3.5 Ct difference with the first time, as if I had my samples diluted 10 times , or I was using the same tubes that the first PCR.
Thinking it was because of the freezing , I conducted a re-dilution range that I kept at 4°C but again same problem , the range behaved as if I had diluted 10 times more. After checking on an another range diluted, I get the same Ct values.

I duplicate my points and one point to another, and only for the standard range, my Ct are very different (but not for my samples, which makes me say that my deposits are fairly uniform, so why I should different Ct for the standard range ?) .

My questions are: is what someone has already been faced with this problem?
How to preserved a DNA plasmid , it is better at 4°C , -20°C? at -80°C?
Knowing that I use Low Binding tubes, is that the plasmid can still bound the tubes ? the cones ?
Is that the same thing would happen with a plasmid extract from bacteria?
What I could do to improve the Ct (I homogenised my tubes by vortex then I go back and forth with the pipette before collecting and once in the wells of the plate I go back and forth with the pipette) ?

Thank you in advance for your answers, sorry for my english, I hope I was clear .

Marie

Hi, i'm not the best at qPCR so I can't really help with that, but I can offer a few tips with using DNA that i've picked up that may help you.  You probably know most of this already, but just in case ..

First i've found that the nanodrop is often inaccurate.  In my lab we call it "the random number generator" because it is generally about 10% inaccurate and can sometimes be as 3x the value out.  I find the most accurate quantification is between 50 and 500 ng/uL, but this is personal experience.

Second, have you tried running your plasmid on a gel after defrosting it?  You don't mention that you use DEPC-treated water or even just autoclaved water, perhaps it is being digested by DNases in water.  Even just autoclaving water before use will greatly reduce RN/DNase activity, though to remove completely its advisable to use DEPC-treated water or nuclease-free water from a commercial supplier.  They generally give loads of it away with kits and I guarantee there will be little 1.5mL screw top bottles of it everywhere in your lab.  Run a gel of fresh reconstituted plasmid and then compare that to a gel of refrosted plasmid.

Third, I find ultra-pure/deionised water generally isn't the best thing to resuspend DNA/RNA in.  You can't use TE for PCR obviously, but i've had great success with 10mM Tris-HCl pH 8.0.  Ultra-pure water is often naturally low pH and can damage your DNA.

Fourth, DNA in nuclease-free water is generally OK at RT for a day, at 4°C for a week ,and at -20°C forever.  -80°C is generally unnecessary.  In Tris/TE it is good for longer, perhaps a week at RT, a couple months at 4°C, and again forever at -20°C.

Fifth, I would be very very surprised to find DNA was binding to low-bind tubes.  I've never really found it to even bind to ordinary tubes.

Sixth, try not to vortex too hard and for too long, as you might damage your plasmid.  Though I was taught this by a very superstitious post-doc so take it with a pinch of salt!  I generally just flick the tube once or twice then pulse spin with a bench top centrifuge.

Scientists working with DNA tend to be a really superstitious bunch so everyone has their own little ways of doing things.  This is just what I do and what i've been taught and I generally do pretty well - though some people may disagree with me! qPCR can be a cruel mistress and sometimes a bit of luck is required.

Good luck.

Hi, 

Thank you for your answer. Since this post, I've tried to run a gel with fresh and refrosted plasmid. The refrosted plasmid was denatured! Actually, this plasmid is not really stable so I've prepared several tube with plasmid at concentration 10^8 cg/10µL and I dilute the standard range in DEPC water for each qPCR. Since, I have not had any problems!

In the laboratory, we think it's a problem of concentration : more there is DNA more it's stable. Even so, sometimes some points of the standard range have a Ct very different from other but, as you said,  sometimes a bit of luck is required! 

DEPC treatment is totally unnecessary for DNA work. DNAse breaks down with heat and vortexing, and is highly sensitive. Also, magnesium is an essential co-factor, so using TE eliminates DNAse activity. I'd STRONGLY recommend using TE (or TE with 0.1 to 0.3 mM EDTA) as your DNA storage medium. Pure water absorbs CO2 from the air, and acidifies. An acid environment breaks down DNA non-enzymatically.  Pure water also does not chelate magnesium ions, so any DNAse present is likely to be active.

You say you can't use TE for your PCR reactions. Why not? typically very very small amounts of template are used so the volume of added TE is quite low. Its major effect will be slightly reducing the magnesium concentration of your PCR reaction. Since PCR reactions typically have 1-2 mM Mg++, adding 5% of a TE solution will reduce that by less than 5%, essentially negligibly.

Your major difficulty with low concentration DNA is almost certainly side wall capture on the tubes. You could consider silanized tubes, and storage in high volume, where the tube area/volume ratio is minimized. For very low concentrations, you probably have to make solutions fresh.