NOTE TO VIEWERS the data plotted must be Ct values versus the LOG of the DNA concentration (or log of the DNA dilution relative to the concentrated standard)
When you dilute a representative DNA sample to make a dilution series for checking the efficiency of a PCR reaction, include dilutions that will include the dilutions you will also use of your actual samples. Note that if you use the slope to calculate Efficiency using just the first more concentrated samples you may get a value that is over 100%. Like 110%. But you cannot actually have more than perfect doubling 100% efficiency. So why do you get >100%? Simple, that is because you have inhibition of PCR in the most concentrated sample and as you dilute the sample the inhibiting factors decrease, which causes the efficiency to increase from its lower, inhibited, efficiency. This gives you a slope and calculation of >100%. You can check the slope using just the more diluted standards and will find the PCR efficiency does not exceed 100%. If possible, dilute your test samples to use the range of dilutions that avoid the inhibited PCR efficiency. It will just take two or three dilutions to often remove this problem. Also, no matter what your dilution is, find out the PCR efficiency around that dilution region and consider using the full equation that includes adding the fractional PCR efficiency for both your gene of interest and normalizer gene PCRs. You can still use the reactions for quantitative analysis even if they are lower efficiency, like 75%, as long as you use calculations that include the efficiency. The simple Delta-Delta Ct method equation is just the simplified form of the equation when the fractional PCR efficiencies are 1 (100%). Ken Mitton, PhD FARVO. Eye Research Institute, Oakland University, Michigan.
Great Video, got to discuss PCR with Breast Cancer Surivor's at San Francisco Avon Walk at Fort Mason. They Love the fact the technology could possibly lead to a cure.
Dear Taqman, I run 3 kDNA PCR with its standard series, when i did the first run the standard was ok, meaning the difference b/n two Ct values was 3.2..... but the run's of standard were inconsistent. so my question was can I calculate the unknown sample by comparing it along with the first st curve. Thank you!!!!
Thank you for your question. A serial dilution is when you start with tube 1 and dilute it into tube 2. You then take tube 2 and dilute it into tube 3 and so forth. The terms dilution series and serial dilution can often be used interchangeable. If you have questions about your experimental setup, please contact us at thermofisher.com/askaquestion. Thank you.
what if my slope is -4.4 should i have to repeat the std curve analysis by making new dilutions or is their any other way to minimize the error in std curve
The slope comes from the equation y = mx + b, which is plotted through the points generated by the efficiency check curve. The points on the x-axis are log [input cDNA], and the y-axis are the Ct values.
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I love how this guy presents the information :) makes me feel joyful
NOTE TO VIEWERS the data plotted must be Ct values versus the LOG of the DNA concentration (or log of the DNA dilution relative to the concentrated standard)
When you dilute a representative DNA sample to make a dilution series for checking the efficiency of a PCR reaction, include dilutions that will include the dilutions you will also use of your actual samples. Note that if you use the slope to calculate Efficiency using just the first more concentrated samples you may get a value that is over 100%. Like 110%. But you cannot actually have more than perfect doubling 100% efficiency. So why do you get >100%? Simple, that is because you have inhibition of PCR in the most concentrated sample and as you dilute the sample the inhibiting factors decrease, which causes the efficiency to increase from its lower, inhibited, efficiency. This gives you a slope and calculation of >100%. You can check the slope using just the more diluted standards and will find the PCR efficiency does not exceed 100%. If possible, dilute your test samples to use the range of dilutions that avoid the inhibited PCR efficiency. It will just take two or three dilutions to often remove this problem. Also, no matter what your dilution is, find out the PCR efficiency around that dilution region and consider using the full equation that includes adding the fractional PCR efficiency for both your gene of interest and normalizer gene PCRs. You can still use the reactions for quantitative analysis even if they are lower efficiency, like 75%, as long as you use calculations that include the efficiency. The simple Delta-Delta Ct method equation is just the simplified form of the equation when the fractional PCR efficiencies are 1 (100%). Ken Mitton, PhD FARVO. Eye Research Institute, Oakland University, Michigan.
Why didn't I find you Videos a year ago!? :(( I would have needed them so bad.
all your vids are very helpful! thanks!
You are a pro! Thanks for the explanation
Great Video, got to discuss PCR with Breast Cancer Surivor's at San Francisco Avon Walk at Fort Mason. They Love the fact the technology could possibly lead to a cure.
Dear Taqman, I run 3 kDNA PCR with its standard series, when i did the first run the standard was ok, meaning the difference b/n two Ct values was 3.2..... but the run's of standard were inconsistent. so my question was can I calculate the unknown sample by comparing it along with the first st curve. Thank you!!!!
Is it possible to import a standard curve from a previous PCR run on the Ab7500? and use it in another assay?
good
what is the difference between dilution series and serial dilution? could you explain how to quantify DNA for forensic human identification? thank you
Thank you for your question.
A serial dilution is when you start with tube 1 and dilute it into tube 2. You then take tube 2 and dilute it into tube 3 and so forth.
The terms dilution series and serial dilution can often be used interchangeable.
If you have questions about your experimental setup, please contact us at thermofisher.com/askaquestion. Thank you.
OMG thx so much for this hint!
what if my slope is -4.4 should i have to repeat the std curve analysis by making new dilutions or is their any other way to minimize the error in std curve
Hi. May I measure the PCR efficiencies of all pairs of primers in one PCR plate? Separately from my unkonw samples.
What is an appropriate method for determining and omitting outliers?
Super, thanks!
What is considered "an acceptable standard material?"
can you please explain the slope value..how its( -3.32)
The slope comes from the equation y = mx + b, which is plotted through the points generated by the efficiency check curve. The points on the x-axis are log [input cDNA], and the y-axis are the Ct values.
What sofware is that at minute 1:09? thanks
That is the StepOnePlus software that comes with the instrument. Hope that helps.
why are you so excited
Because Science!