How To: PCR Calculations

How To: PCR Calculations


Hello, my name is Sunil. I work for the life science business of Merck KGaA, Darmstadt, Germany. I’m an RTS and I live in New Jersey, USA. In this video tutorial, I’m going to walk
you through the mathematical calculation that’s involved in setting up a PCR, the preparation of the Mastermix, and also scaling up those values when setting up multiple PCRs at the same time. So in this example, let’s say that we have extracted genomic DNA from four separate strains of E. coli. And we want to clone, we want to detect gene X, in all the four strains. So we are going to set up a total of four PCRs. The reaction volume for each PCR is going to be 25 microliters. Okay. So let’s take a look at some of the math. Each PCR tube is going to have each of these components. These components have been stored as working stocks, the concentrations of which you can see in this column over here. So we have a 10 millimolar stock of the dNTP Mix. We have a 10x PCR Buffer. We have the forward and reverse primers as 10 micromolar working stocks. We have the magnesium chloride at 25 millimolars and the Taq polymerase at 5 units per microliter. This column over here shows you the final concentrations that I want each of these components to be at in an individual PCR tube. So what I need to determine is, what volumes do I take of these working stocks and add to an individual PCR tube? So for that, you calculate the dilution factor. Let’s do that for the first ingredient, the dNTP mix. We have a 10 millimolar working stock. The final desired concentration is 0.2, so if you take the 10 millimolar stock, divide it by the desired concentration of 0.2, that gives you a dilution factor of 50. We know here that every individual PCR is going to have a final reaction volume of 25 microliters, that’s what we’ve decided. So take that 25 microliters, divide it by the dilution factor of 50, and that gives you your volume of 0.5 microliters. So what this tells us is, I need to take 0.5 microliters of the 10 millimolar stock of the dNTP mix in my final reaction volume of 25 microliters for an individual PCR tube and that will bring it down to the desired concentration of 0.2 millimolars. I have used the same logic to calculate the volumes for each of the remaining ingredients and that’s shown over here in this column. So this is a column for a single PCR tube
of 25 microliters. Now another thing to take into consideration is every, all four PCR reactions have these ingredients in common, except the template DNA. That’s the only thing that is unique to all the four reactions. So it would be very cumbersome, very inefficient for me, to take each of these components and add them to the four PCR tubes. It makes more sense for me to make a bulk Mastermix for all the four reactions together, adding each of these components. The way you do that calculation… this column over here shows you the volumes for a single PCR tube. You multiply that by the total number of PCRs that you’re conducting, which is four. You will notice , and that’s this column over here, the last column, you will however notice that I have actually done the calculation
for five reactions instead of four. The reason I have accounted for an additional reaction is to make up for any errors that could occur during pipetting. These are the volumes for each of these ingredients over here. This will give you a volume of 120 microliters. This is your bulk Mastermix for five reactions. You’ll mix everything evenly and then you will aliquot 24 microliters from this bulk Mastermix into each of the four PCR reaction tubes. The only thing you have left to do now is to add a microliter of the template DNA from each of the four equalized strains into the four tubes. That gives you your four tubes, each with the required reaction volume of 25 microliters. They’re ready to go into the thermocycler and you’re ready to start your PCR. Hope you found this helpful. Thank you for your time. Goodbye.

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