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Graphs
As I have previously mentioned, in terms of analyzing for efficiency, I will be looking at: 1. The amount of product formed in a given time (the minimal number of cycles/time for product formation) – analyzed with Ct. values. 2. How well PCR product formation adheres to the theoretical exponential growth – analyzed with product-formation graphs. The included graphs above contain data collected from the real-time PCR – the graphs display the growth of amplicon/PCR product during the reaction using the 7 dilutions and the No Template Control. GENERAL EFFICIENCY Generally we can see that the Ct. values decrease when we increase the magnesium chloride concentration; this tells us that the MgCl2 concentration has some influence upon efficient initial PCR product formation. EFFICIENT PRODUCT FORMATION (IN TERMS OF TIME) To find the optimal concentration that is efficient for product formation, we look at the preceding Ct. value of the one that no longer changes or that increases (in this way, it is said that further magnesium chloride manipulation will no longer improve efficiency). There is only one set of values that have little change (dilution 10-1, 3.5-4.0mM) – so we have to look at Ct. values that increase instead. Looking at the graph, we can see that the Ct values generally decrease and after 3.5mM, the Ct. value increases (this is generally towards the more diluted DNA samples, from 10-2 to 10-6). However, ignoring that aspect and looking at the 100 dilution it is suggested that a concentration greater than 4.0mM may be the ideal for efficient initial product formation since it does not yet show a decline in required cycles. EFFICIENT THEORETICAL EXPONENTIAL GROWTH Unfortunately, due to time constraints, I was unable to calculate and create a standard graph of fluorescent growth; such a graph would display a growth pattern of a 100% efficient reaction (products that continually grow exponentially). Had I constructed such a standard graph, I would have compared it to the shapes of the resultant graphs. Or, to be more accurate, use the numerical data of a standard fluorescent graph and compare it to the numerical data from each of the four magnesium chloride concentrations. A deviation from the standard exponential growth would show that the particular magnesium chloride concentration was not 100% efficient. To find the optimal magnesium chloride concentration I would look at the graph that displays data closest to the standard fluorescent graph. The second method of comparing to the theoretical growth involves a “standards graph” – this involves creating a logarithmic graph of a known number of DNA copies (a number that parallels an exponential amplification) versus the Ct value [see appendix D for an example]. The results are then plotted onto the same graph and should align along the generated line – if the line adheres to a slope of -3.2 to -3.4 the results can be considered efficient. Like the fluorescent standard graph, we can be more precise by looking at the numerical values for comparison – the concentration with values closest to forming a -3.2 to -3.4 sloped line is the optimal. We can rule out contamination in affecting the results since the no template control amplified DNA. If the graph showed an amplification of the no template control – it would mean there was DNA present and thus, there was contamination. Back to Top | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||