How to set up a PCR

the polymerase chain reaction or PCR was first described over 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 quantity of DNA template even down to just a single DNA molecule PCR amplification means that detection and identification of your target DNA can be made using visual techniques based on the size and charge of the piece of DNA you have amplified the theory behind every PCR whatever your DNA target is the same a mixture is created that contains five basic things your DNA template a DNA polymerase enzyme forward and reverse primers and 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 or near your region of interest usually they are between 20 to 30 bases or nucleotides long the enzyme is there to extend the primers which have annealed 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 should only anneal to this particular region of interest during the 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 the polymerase can begin the initial heating step is called denaturation and usually occurs at 94 or 95 degrees centigrade it needs to be at this temperature long enough to denature the template completely the following step when the primers are able to bind to the template at a lower temperature is called annealing this temperature depends on the TM or melting temperature of your specific primers and is usually between 50 to 65 degrees centigrade this step doesn't need to last very long typically 10 to 30 seconds the final step in this first amplification cycle is the extension at 72 degrees centigrade this is the optimum temperature for the DNA polymerase to build the complementary strand following on from the three prime end 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 on the length of your target but 30 seconds to 1 minute is usually long enough for most products this process of denaturing annealing and extension is repeated 30 to 40 times theoretically doubling the number of DNA copies at each cycle and thereby increasing exponentially the number of copies of your target of interest in the mixture 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 instrument the lab area used to set up and run your reactions needs to be carefully controlled and maintained PCR can detect a single molecule of DNA so it is essential to have a unidirectional workflow with ideally three separate rooms in your system firstly 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 into the area where PCR products are generated or analyzed the best system would involve a workflow where the cleanroom would not be revisited on a day when you've been in other potentially PCR product contaminated areas the second room in your system would be the extraction lab where you've prepared what a template ready to put into the PCR once you've set up your reaction in your cleanroom you would take the reaction tubes or plates to the extraction lab and add your DNA template from there you can go on to the third room the PCR machine room this is where the PCR s take place and where the products are usually analyzed by visualization on a gel or by other methods it is best to consider that PCR products that you generate contaminate this third laboratory however careful you are so never setup a PCR reaction here or take reagents or consumables from this lab into the other rooms or they could contaminate your clean system the proportions of the reaction mixture have a great influence on the quality of PCR results there is a general formula for concentrations of template enzyme primers and nucleotides to use but this can be varied a little if necessary the general guidelines are naught point five to two units of TAC polymerase 200 micro molar of each dntp not point two to naught point five micro molar of each primer 50 to 500 nanograms of genomic template and one times buffer conveniently there are also PCR master mixes available with all the components combined for you so all you need to add are your primers and template today we are going to use the clean system to set up six reactions three PCR s and three control reactions one negative control one positive control and finally an extraction control always use negative and positive control reactions to ensure the results are due to amplification of the right sample to make sure we have enough reaction mix to go round we will make up enough for seven reactions the six reactions already mentioned plus one extra in the clean room set up your reactions on ice with a master mix you 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 micro liter reaction use 25 microliters of master mix per reaction for our seven reactions today we will use seven lots of 25 microliters which 175 microliters in total then add seven microliters of each of your primers which have been diluted to 10 micromolar or 10 peak moles per microliter that's one microliter of each primer for every one of your seven reactions usually one microliter of DNA template would be sufficient for each reaction so make up your reaction mix to 333 microliters with water supplied with the master mix this volume is calculated by subtracting the remaining volume to be added to the final total volume of 340 microliters dispensed 49 microliters of this mix into seven labelled 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 you leave in 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 positive control and a positive control that has been taken through your extraction procedure to control for inhibition close the tubes remove your lab coat then take the tubes to the PCRM place the tubes into the instrument and start the cycling there are lots of different instruments you can use today we've used a block-based instrument also available are real time instruments that tend to be faster than block-based ones and measure fluorescence in real time as pcr products are generated you

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Keywords: polymerase chain reaction, PCR, DNA, primers, denaturation, annealing, extension, amplification, nucleotides, DNA polymerase enzyme, buffer

Id: V2JYy6-DE9c

Length: 8min 22sec (502 seconds)

Published: Thu Jan 17 2013