Question

I need a full discussion page explaining the results of this experiment based on the graph provided was the experiments successful? Explain each line on the graph. what they mean and why could be the reason they cross-link?w Objective In the following experiment, we will use your recently isolated gDNA to set up preliminary qPCR reactions. Because mitochondrial DNA should be in higher concentration in your DNA samples, and qPCR performs better with products of low molecular weight (small size DNA molecules, around 200 bp is ideal, 500 bp is the max.), we will use the human mitochondrial primers. This primer set was used in your A reaction and is designed to amplify a 400bp product. To further explore the range of possibilities associated with qPCR, such as relative quantification of gDNA, you will prepare multiple dilutions of your DNA samples. Background Real time PCR relies on the use of fluorescent markers that are designed to detect the synthesis of double stranded DNA (dsDNA) in real time. The one that we will use today is based on the SYBR Green I dyc. This chemical compound can not only bind to dsDNA, but also fluoresces when it is bound. When the DNA denatures, the SYBR Green I dye is released and the amount of fluorescence that can be detected is dramatically decreased. 1. Set up a scrial dilution of your DNA samplc. We will test 10X and 100X dilutions, in order to illustrate how little DNA is needed in these types of reactions. Take two 500 ul tubes and place 9 ul of H20 in them. Pipet 1 ul of your DNA in the first tube, pipet up and down, then pipet 1 ul of the mix and place it in the second tube. 2. Set up a Master Mix for your PCR reactions. Every reaction will occur in a 25 ul volume. Most of the PCR "ingredients" are already combined in a mix labeled "Reaction Mix", which typically needs to be diluted by 2 (2X concentrated). Part of the dilution process includes adding the primers and gDNA template Since you will set-up 4 reactions, you can prepare a Master Mix that will contain enough material for 4 reactions. The Master Mix should contain all the reagents that are common to all 4 reactions. 3. Divide your Master Mix into four tubes using the 8-tube strip provided for you. All tubes in the strip should reccive an cqual amount of Master Mix (96/4-24 ul) 4. Add 1 ul of each of your gDNA templates into each tube. Your four reactions will contain the following template: lul of your original DNA sample (no dilution) 1 ul of a 10-fold dilution of your gDNA sample lul of a 100-fold dilution of your gDNA sample 1 ul of H20 (no template control, or NTC) Results 0.600 Negpkve 0.500 No dl 0.300 0.200 Diluted 0100 diluted χ10 0.000 15 20 25 30 35 SYBR Green

Answer 1

Well ! You have 4 samples and 4 lines in your graph. The X axis corresponds to the no of cycle in your qPCR and Y axis corresponds to fluorescence intensity. But, in this case, you can say that your experiment is not successful. Why? Look at the line that corresponds to no dilution. In this case, you are getting a good fluorescence at cycle 20. That means the concentrated DNA is amplified well after 20 cycles. That's great. Now you look at the line that corresponds to 10X dilution. After 20 cycles of amplification, it is showing a fluorescence (more than 0.1) that is less than no dilution sample (0.2). So, this is ok. But now you look at the line corresponds to 100X dilution. Are you getting a significant difference between 10X and 100X dilution? No. In fact after 20 cycles of DNA replication, fluorescence is almost same (more than 0.1) in both the cases. Do you think it should happen? The answer is absolutely no. Because the difference in DNA concentration will reflect the difference in fluorescence. That's why this is qPCR. Because the fluorescence is quantifiable and we should obtain a significant different fluorescence intensity in two different dilutions. The last thing is negative control. Look at the line that corresponds to negative control. Do you think that you should get any fluorescence when you have no DNA at all in your sample? So, what's about this. In your negative control sample, you dont have any DNA. That's why you used this as a negative control. But look at the graph. You are getting a good amount of fluorescence after 25 PCR cycles. This is absolutely wrong. Either you have DNA contamination in your negative control sample, or there are a problem with primer dimer. Thus, you are getting a wrong but huge fluorescence, even more than no dilution sample in your negative control. So, the experiment is not at all successful.