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qPCR amplification interference fixed with freezing


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#1 TIMESUP

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Posted 11 April 2011 - 03:10 PM

Hello All,

Attached are two amplification graphs I've developed using an Applied Biosystems StepOnePlus PCR machine. The orange and blue curves are my samples. The black curve is my positive control. The Ct value is set to 0.05. My template is DNA.

The results in the first graph were obtained by running the samples right after I finished their extraction. The results in the second graph were obtained by running the same samples after they were frozen at -20C for over 24 hours. As you can, see the slopes of the curves of my samples differ before and after being frozen. Normally my samples have a similarily sloped curve to the positive control curve.

This issue suddenly occured seemingly out of nowhere a few weeks ago. I have not made any changes to buffers or protocol. It seems there is something in my extracted samples which is inhibiting amplification, but is removed after freezing for 24 hours.

Any comments will be appreciated!

Attached Thumbnails

  • before freeze.jpg
  • after freeze.jpg


#2 Trof

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Posted 12 April 2011 - 12:10 AM

I personally wouldn't make a theory from single occasion. Your first reaction may have been screwed in some way, even if you think you did everything the same way. That happens, you repeat it and it's suddenly OK. There are many things that could happen, like improper dissolving of the isolate. Freeze-induced degradation of inhibitor would be pretty low on my list. IMHO.

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#3 TIMESUP

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Posted 12 April 2011 - 07:38 AM

I have actually observed this issue with approximately 20 different samples extracted across 5 days. Each time my results from the initial run have shallow curves. The next day I rerun them after being frozen and the curves are normal.

#4 Trof

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Posted 12 April 2011 - 10:54 AM

That's more interesting.
Still, dissolving may be the issue. Did you try to leave some in the fridge and not freezing them, and repeat next day?

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 TIMESUP

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Posted 12 April 2011 - 11:32 AM

I split up 2 extracted samples into 5 aliquots and ran them under these conditions:

1. As-is
2. 1 hour freeze
3. 24 hour room temperature
4. 24 hour fridge
5. 24 hour freeze

The 1 hour freeze curve was a little better than the as-is, but still too shallow. The 24 hour freeze curve was normal. The 24 hour fridge curve was a little better than the 24 hour room temperture curve. The curves between the 24 hour freeze, fridge and room temperature did not differ greatly, but they were more normal with the decrease in temperature.

#6 Maddie

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Posted 15 April 2011 - 08:29 AM

I split up 2 extracted samples into 5 aliquots and ran them under these conditions:

1. As-is
2. 1 hour freeze
3. 24 hour room temperature
4. 24 hour fridge
5. 24 hour freeze

The 1 hour freeze curve was a little better than the as-is, but still too shallow. The 24 hour freeze curve was normal. The 24 hour fridge curve was a little better than the 24 hour room temperture curve. The curves between the 24 hour freeze, fridge and room temperature did not differ greatly, but they were more normal with the decrease in temperature.



So the 24h in the fridge was the best, right?
When we extract, we store the extracts in the fridge if we know we are going to work on them the coming days (or sometimes weeks). Then we store them in the freezer. Freeze/thaw events sometimes slightly degrade DNA.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#7 TIMESUP

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Posted 18 April 2011 - 07:56 AM

So the 24h in the fridge was the best, right?
When we extract, we store the extracts in the fridge if we know we are going to work on them the coming days (or sometimes weeks). Then we store them in the freezer. Freeze/thaw events sometimes slightly degrade DNA.

The 24h fridge and freezer results were very close.

I'll take this into consideration and run some temperature experiments to see how sensitive my extracted DNA is. The person who gave us our extraction and amplification protocols told us to freeze samples that won't be amplified within 24 hours.

Thank you Maddie and Trof for your input.




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