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pcr product quantification


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#1 deganuti

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Posted 15 July 2009 - 11:34 AM

Hi friends,

I have some doubts about my the quantification of pcr products using a nanodrop spectrophotometer:
1- what is the best solvent to blank the measurement: the buffer (pcr buffer, dntps, primers, water, without enzyme) ou just water pure? How the dntps or primers can change the measurements?
2- in the first experiments, the quantification was ok (spectrophotometer x agarose gel), and I did a measurement in nanodrop using pcr buffer. But now, the measurement using pcr buffer shows only negative values for dna concentration.
Someone can help me?

Thanks in advance.
Dega

#2 krisztina

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Posted 15 July 2009 - 02:23 PM

Hi Dega,

Just a couple of thoughts, before an expert turns up and helps you out. I have no personal experience on this.
Why don't you try water as setting the blank and measure your buffer as well. That'll tell you if there is a difference between water and buffer (likely that there is, but I probably not significant). The negative values can derive from contamination of your buffer for instance. Also, after the pcr run, the conditions in the reaction are not exactly the same as before the run, therefore the buffer might not represent the exact conditions in your reaction.

Best of luck



Hi friends,

I have some doubts about my the quantification of pcr products using a nanodrop spectrophotometer:
1- what is the best solvent to blank the measurement: the buffer (pcr buffer, dntps, primers, water, without enzyme) ou just water pure? How the dntps or primers can change the measurements?
2- in the first experiments, the quantification was ok (spectrophotometer x agarose gel), and I did a measurement in nanodrop using pcr buffer. But now, the measurement using pcr buffer shows only negative values for dna concentration.
Someone can help me?

Thanks in advance.
Dega



#3 jiajia1987

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Posted 15 July 2009 - 05:27 PM

Hi deganuti,

I am not too sure how others do it, but I will just tell you my way of quantifying my samples with nanodrop.

After my PCR, I will always do a cleanup before I do any quantification with nanodrop. I always elute my samples with water, so I will use the water alone as a blank. I am not too sure if you need to use the buffer as a blank and you used the buffer for elution.

#4 ram

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Posted 30 July 2009 - 07:30 AM

I feel same as jiajia1987...
You are quantifying your PCR product meaning you are going to use it for some downstream application (can u explain?). If so, you most probably have to purify the product. Why don't you do this first, so that you get rid-off so many components from your actual PCR product. And then you can use water/elution buffer whichever is used for eluting the product, as a blank.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#5 phage434

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Posted 01 August 2009 - 05:50 AM

It is essential to purify the PCR product prior to quantification. The PCR reaction has huge amounts of primer and dNTPs present, which behave with respect to UV absorbance identically to long chains of DNA. Unless you purify the DNA product, you will see no difference in the measurement before or after the PCR reaction.

#6 ph1ll1ps

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Posted 11 August 2009 - 06:45 AM

hi,

to add something. The PCR buffer contains lots of salts (sometimes also NTP's) that absorb at 230nm, this will have a major inpact on the 260nm absorbance of your DNA peak, so don't use the PCR buffer for blanking.




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