Thank you very much, efficiency calculation really helped me out! Your method is different from my profs... I tried using his and was getting efficiency of over 3000%, using your method, the numbers were much more realistic, 93%
how is your Ct value so low when you have highest dilution? (low Ct values means high initial concentration of DNA, and at higher dilution how can you have higher initial concentration?)
Hi Vikas, It's DNA Copies/mL plotted against Ct value. DNA concentration will be high in lower dilution and as we dilute more no of DNA molecule will be less. Please note the plot is done in reverse meaning that the low no of DNA Copies has high Ct value (the highest diluted sample). Hope it is clear now.
Not sure I can quite follow what you want to say? And how could your Ct Value is so high, while your dilution/Copy number is very low? - Low dilution >>> low Ct value>>> high copy number. - High dilution >>> High Ct Value >>> less copy number. Could you please clarify it a bit further? Thanking you !
Hi OMAR, Here is the explanation: If you start with 1,00,000 copies/mL of DNA and assume you got 16Ct, here the Ct value is low in number and the copy number is high. Once you perform dilutions of the same sample, let's say another 4 serial dilutions of ten fold , your Ct will shift to ~30, which is high number and the copy number will be less in that sample since we have diluted it. Note: Each 10 fold dilution will shift the Ct by ~3.32, in a well optimized assay. Hope this helps.
Hi, Ideal PCR efficiency for a well optimized assay is between 90 - 105%. You should try to get the assay efficiency in that range. Please see below link on resolving issues with PCR efficiency. th-cam.com/video/H8aP7AWlPa0/w-d-xo.html
I have a question, if the mean of my ct value is 27.6. When I plug this number into my standard curve equation to generate DNA concentration, the concentration is like 660, which doesn't line up with my standard curve plot.
simply explained without speaking, well done!
thanks u so much for the video, it helped me a lot in preparing my lab report ❤
How do I know the copy number of the cDNA? I just did a serial dilutions. How to fit the dilution levels to the plot?
You need to the concentration of your RNA. Rate of RNA to cDNA conversion is 1:1. So, x amount of RNA would generate x amount of cDNA.
Thank you very much, efficiency calculation really helped me out! Your method is different from my profs... I tried using his and was getting efficiency of over 3000%, using your method, the numbers were much more realistic, 93%
Excellent video, extremely helpful.
How Can I calculate unknown ct value copy number through this equation ?
how is your Ct value so low when you have highest dilution? (low Ct values means high initial concentration of DNA, and at higher dilution how can you have higher initial concentration?)
Hi Vikas,
It's DNA Copies/mL plotted against Ct value. DNA concentration will be high in lower dilution and as we dilute more no of DNA molecule will be less.
Please note the plot is done in reverse meaning that the low no of DNA Copies has high Ct value (the highest diluted sample).
Hope it is clear now.
@@BioResource thank you so much for replying, and I could note that the plot is done in reverse meaning... thanks for clarification
Not sure I can quite follow what you want to say? And how could your Ct Value is so high, while your dilution/Copy number is very low?
- Low dilution >>> low Ct value>>> high copy number.
- High dilution >>> High Ct Value >>> less copy number.
Could you please clarify it a bit further?
Thanking you !
Hi OMAR,
Here is the explanation:
If you start with 1,00,000 copies/mL of DNA and assume you got 16Ct, here the Ct value is low in number and the copy number is high. Once you perform dilutions of the same sample, let's say another 4 serial dilutions of ten fold , your Ct will shift to ~30, which is high number and the copy number will be less in that sample since we have diluted it.
Note: Each 10 fold dilution will shift the Ct by ~3.32, in a well optimized assay.
Hope this helps.
@@BioResource Got you! Thanks so much for your update.
i did the standard curve as u did following every step and I got the pcr efficiency 110% is it realistic
Hi,
Ideal PCR efficiency for a well optimized assay is between 90 - 105%. You should try to get the assay efficiency in that range. Please see below link on resolving issues with PCR efficiency.
th-cam.com/video/H8aP7AWlPa0/w-d-xo.html
Copies/mL? Not copies/uL (microlitre)?
Here it is copies/mL. Can be expressed in copies/uL also.
Hello, I am trying to develop your method, but I can´t because I have a low slope and I don´t know how to solve it. Thank you!
+Mercedes Jiménez May be you have to relook into assay conditions to get the efficiency between 90 - 105%.
I have a question, if the mean of my ct value is 27.6. When I plug this number into my standard curve equation to generate DNA concentration, the concentration is like 660, which doesn't line up with my standard curve plot.
Could you please share how you did the calculation?
For anyone here with the same question, you can't just plug your Ct value into X, your Ct value is y so you need to solve for X.
pretty cool! Thank you so much!
Thank you. Consider subscribing 😊
great help!!
This is great, thank you!
Glad it was helpful.