How to set up a PCRDec 28, 2021
The polymerase chain reaction or PCR was first described more than 20 years ago and is a technique for making millions of copies of the gene or a particular DNA target of interest. Copies are made using a very small initial amount of DNA template, even just one. PCR amplification of DNA molecules means that detection and identification of your target DNA can be done using visual techniques based on the size and charge of the piece of DNA you have amplified. The theory behind every PCR, whatever its DNA target, is the same. contains five basic things your DNA template an enzyme DNA polymerase primers and forward and reverse nucleotides also known as dntps and buffer to stabilize the enzyme for each new PCR you choose the primers and design them to be complementary to short sections of DNA next to them or close Your region of interest is usually between 20 and 30 bases or nucleotides long.
The enzyme is there to extend the primers that have hybridized to a specific region of your template and use the free nucleotides in the mixture to build the new strand complementary to your target. The primers only need to hybridize to this particular region of interest during PCR, the DNA being amplified is heated and the double strands separate upon cooling, the primers bind to the template and the process of adding complementary nucleotides by polymerase can begin. The initial heating step is called denaturation and usually occurs at 94 or 95 degrees Celsius. It is necessary to be at this temperature long enough to completely denature the template.
The next step, when the primers can bind to the template at a lower temperature, is called annealing. This temperature depends on the temperature. TM or melting temperature of your specific primers and is generally between 50 and 65 degrees Celsius. This step doesn't need to last long, usually 10 to 30 seconds. The final step in this first amplification cycle is the extension to 72 degrees Celsius. This is the optimal temperature for the DNA polymerase to build the complementary strand from the three main ends of the primer using the free nucleotides, the polymerase adds them at around 50 to 100 bases per second, so the duration of this step will depend of your target length, but 30 seconds to 1 minute is usually sufficient for most products.
This process of denaturation, annealing and extension is repeated 30 to 40 times, theoretically doubling the number of DNA copies in each cycle and therefore exponentially increasing the number of copies of your target of interest. In mixing, samples can be prepared and incubated in a thermal cycler and a complete PCR reaction can be performed in a few hours or even less than an hour with certain types of rapid instruments. The laboratory area used to set up and run your reactions must be carefully controlled and maintained. PCR can detect a single DNA molecule, so it is essential to have a one-way workflow with, ideally, three separate rooms in your system.
First of all, you must have a clean room. This room would contain all of the reagents for your PCR except the DNA template: none of the reagents and consumables would have been in the area where the PCR products are generated or analyzed. The best system would involve a workflow in which the clean room would not be revisited on a day when it had been in other areas potentially contaminated with PCR products. The second room in your system would be the extraction lab where you prepared a template ready to place in the PCR. Once you have your reaction set up in your clean room, you would take the reaction tubes or plates to the extraction lab and add them. your DNA template from there can go to the third room, the PCR machine room, this is where PCRs are performed and where the products are usually analyzed by visualization on a gel or by other methods.
It is best to consider that the PCR products you generate will contaminate this third laboratory no matter how careful you are, so never set up a PCR reaction here or carry reagents or consumables from this laboratory to other rooms or they could contaminate your clean system. The proportions of the reaction mixture have a large influence on the quality of PCR results: there is a general formula for the concentrations of primers and template enzyme nucleotides to use, but this can be varied slightly if necessary. General guidelines are zero point five to two TAC polymerase units. 200 micromolars of each dntp, not even point two to zero. point five micromolars of each primer 50 to 500 nanograms of genomic template and a buffer once Conveniently, there are also PCR master mixes available with all the components combined for you, so all you need to add are the primers and template you today we are going to use the clean system to set up six reactions three PCR and three control reactions a negative control a positive control and finally an extraction control always use negative and positive control reactions to ensure that the results are due to amplification of the correct sample to ensure we have enough reaction mix for everyone we will prepare enough for seven reactions the six reactions already mentioned plus an additional one in the clean room set up your reactions on ice with a master mix use half the total volume of your PCR reaction usually 10 to 50 microliters and then make up the total volume with your other reagents, so for a 50 microliter reaction use 25 microliters of master mix per reaction.
For our seven reactions today, we will use seven batches of 25 microliters, which is 175 microliters in total, then we will add seven microliters of each of their primers that have been diluted to 10 micromolar or 10 peak moles per microliter, that is, one microliter of each primer for each of its seven reactions. Typically one microliter of DNA template would be sufficient for each reaction, so prepare your reaction mixture to 333 microliters with water supplied with the master mix, this volume is calculated by subtracting the remaining volume to be added from the final total volume. 340 microliters. Dispense 49 microliters of this mixture into seven labeled tubes that will fit your PCR machine.
Now take the tube to the extraction room to add your template DNA remember to change your lab coat before leaving for the extraction lab add one microliter of your template DNA or control material use water for a negative control a known positive extract for your control positive and a positive control that has The extraction procedure has been performed to control inhibition. Close the tubes. Take off your lab coat. Then take the tubes to the PCRM. Place the tubes into the instrument and begin the cycle. There are many different instruments you can use today. We have used a block.
Real-time block-based instruments are also available, which tend to be faster than block-based instruments and measure fluorescence in real time as PCR products are generated.
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