What is the blank used in this case? I want to know the concentration of DNA after PCR run. Can I simply use nanodrop or other component of mastermix would interfere with my result?
NO NO NO. The primers and nucleotides also absorb UV light!! Thus, Nanodrop will measure everything in the PCR mix, not only your PCR product but also primers, probably template DNA (could be relevant if there are larger amounts of gDNA) and even free dNTPs. You MUST purify the PCR mix before measureing the conc. of PCR products. And then you use the solvent (e.g. water or TE) as blank.
Quantitative gel electrophoresis, using known concentrations of DNA standards, is a better way to measure DNA post-PCR. I wouldnt advise you to use nanodrop.
Nano Drop spectrophotometer is a good option for estimation of DNA concentration. Nuclease free water can be used as blank.
Yes you can use spectrophotometer to measure your DNA concentration. Its best that you use water or your pcr buffer as blank.
In my opinion, for the quantification with nanodrop, you should use the elution buffer or water in which you have eluted your DNA sample if you are using purified PCR product after PCR. if it is not the case, you want to make estimation directly after PCR run then you should use the PCR mix which you have used for PCR (PCR mix plus the template for accurate estimation).
I think it will help you.
NO NO NO. The primers and nucleotides also absorb UV light!! Thus, Nanodrop will measure everything in the PCR mix, not only your PCR product but also primers, probably template DNA (could be relevant if there are larger amounts of gDNA) and even free dNTPs. You MUST purify the PCR mix before measureing the conc. of PCR products. And then you use the solvent (e.g. water or TE) as blank.
As Jochen had said: NO!
Everything else in the mixture will absorbe UV light, even if you use the PCR mixture before the reaction as a blank, you will not get a useful result.
Hi, not agreed with above comments, just acknowledge Wilhelm comment, don't ever measure DNA concentration directly after PCR because now here DNA with all other dNTPs, primers which have there on absorbence , measured DNA conc. before PCR and if want to measure after PCR then must purify product then use water as control.
I would suggest you to run your sample on gel. Not only would you able to tell the difference in DNA concentration due to different intensity of the band. You would also be able to do DNA gel extraction and get much much better amplified target following gel extraction protocol. If desired, you then can use pico green dye in couple with Spectrophotometer / nano drop to quantify your products.
Dear drs
If I use pcr mastermix as bufferi think I would able to remove interference due to dNTP and primer???
Nanodrop is not the perfect way to measure DNA conc. after PCR. You can use quantification DNA ladder. best of luck
Yes, the concentration of the DNA before and after PCR is different., expect higher concentration after PCR run due to some proteins and impurities.
Qubit (Invitrogen) is also the best instrument to measure the actual concentration of DNA either before or after PCR run.
@Shaimaa: the PCR mix after a PCR is not similar to the unused PCR mix. After the PCR, primer and dNTP concentrations are lower, and there is an accumulation of pyrophospates (however, I don't know if they absorb at 260nm, but it is a difference to the untouched PCR mastermix). So using a new mastermix as blank does not really work here. And even if this would not be a problem: the relatively high absorbance of primers and dNTPs would impede an accurate quantification of the PCR product (the higher the "background"-absorbance, the lower the signal-to-noise ratio).
Need to purify your samples before you try to quantify for several reasons,
as stated by others;
-the original template DNA, primers and dNTPs will allow false positives and cause you to overestimate the concentration of your actual product.
- No matter how confident in your primers and annealing temperatures, you cannot be sure that all the DNA amplified is the product you want, it is not uncommon for multiple sized products to be amplified in a PCR reaction and therefore you cannot even trust that a product after PCR purification can be confidently quantified without running it on a gel.
My advise, always run the product on a gel first. You can determine relative concentration pf product by comparing the UV intensity to a good DNA ladder. Check to see that you only have one band (which is separate from the primer-dimer and even from any of the template). then purify the product either from the gel or from the original PCR mix, and then quantify using a nano-drop.
It may seem like a waste of an hour to run a gel but it can save much more time and confusion in the long run if the aforementioned problems occur.
As stated by others, you would be better off if you use nano drop after purification for the many obvious reasons. However, if you still want to determine the conc as accurate as possible immediately after PCR, may I suggest for you to run your sample on the bioanalyzer. It can provide rather accurate conc of the specific band of your amplification.
If it is a purified sample I would measure it on a Trinean instrument. They have two instruments, that allow quantification without any dye.
Yes, you can but not only the DNA but also other proteins and impurities will be measured.
The best tool is Qubit that measures specific targets such as DNA, RNA, or Protein.
Good luck
Nop. With the Trinean instruments they do quantification and qualification. Meaning that they will tell you the impurities just based on algorithms. Their results for what I saw so far are really close 1-5% from Qubit. And they don't use any dyes.
www.trinean.com check their website. If it's still updated they have all the information there.
What I did mostly, after the PCR product purified I used DFW or UPW to measure the concentration of the DNA by Nanodrop and It works well.
Wouldn't it be possible to use a control sample that you did not run in the PCR-machine as zero-control? Than it would just for the template if expected to be in the same concentration.
I wonder, does the Nanodrop measure dNTPs that are not incorporated also?
Angelica, yes, you measure the absorption of UV-Light, and nucleotides do absorb UV light. The spectormete can not distinguish if the light was absorbed by free nucleotides of be nucleotides being part of a polymer.
The absorption is due to the aromatic electron systems in the bases, and these don't change upon polymerization. In polymers you will have stacking effects that have some impact on the absorption (because the pi-electron systems of stacked bases influence each other), and this is in fact measurable, but the effect is small compared to the effect of having nucleotides in the solution at all.
I completely agree with Jochen Wilhelm, the dNTPs, primes, dimers .....etc can make wrong measurements as they absorbs UV like PCR product. Actually I faced this problem in the past. I think the best way to measure the concentration of PCR product can summarized by purification-elution with DDW or TE then measurement. Thanks for this wonderful discussion.
Is it possible to measure the PCR products without purification with Qubit?
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