The overall goal of this procedure is to demonstrate the steps involved in the polymerase chain reaction or PCR, which is used to produce an ample supply of a specific segment of DNA from a small amount of starting material. First, assemble the reagents and materials to be used in the PCR reaction, then set up the reaction mixture, including the four deoxy ribonucleotides, the DNA template, the primers, and the DNA polymerase. Next program, the thermal cycling conditions and run the PCR reaction.
Following this. Check the results to determine if the reaction was successful. Ultimately, a polymerase chain reaction should yield a specific amplicon of the desired product size.
Generally, individuals new to this protocol will struggle a little bit with setting up the calculations of each of the variable reagents, and sometimes they have troubles with the pipetting, the correct volumes, especially with the polymerase which has glycerol. Begin the experiment with a freshly filled ice bucket. Wear gloves to avoid contaminating the reaction.
Mixture and reagents. Arrange the PCR components on ice to completely thaw. These include the DNA template primers, DNA polymerase 10 x reaction buffer with or without magnesium chloride, deoxy nucleotides, sterile water and magnesium chloride.
The magnesium chloride is required if using the buffer without magnesium chloride. Place a 96 well plate into an ice bucket as a holder for the 0.2 milliliter thin walled PCR tubes. Now equip the workbench with PCR tubes and caps.
A PCR tube rack, an ethanol resistant marker, and a set of micro pipetters. Always create a table of reagents detailing the reaction mixture with quantum sterile distilled water to obtain final volume of 50 microliters. For example, using five microliters of a buffer without magnesium, one microliter of 10 millimolar DN NTPs, and an optimized 4.0 millimolar magnesium.
One microliter of each 20 micromolar primers, 0.5 microliters of a two nanograms per microliter template. And 0.5 microliters of polymerase would require 33 microliters of sterile water only. Add magnesium chloride if it is not present in the 10 x buffer or if needed for PCR optimization.
Proceed to label the PCR tubes with an ethanol resistant marker to each reaction tube. First, add the calculated amounts of sterile water, then add 10 x buffer and 10 millimolar DN NTPs. For this reaction, we are performing a titration with magnesium chloride.
Since the magnesium concentration will not be constant, magnesium chloride is added to the PCR tubes individually, and the volume is normalized to 10 microliters with sterile water, such that 40 microliters of the final master mix is added to each PCR tube. To complete the 50 microliter reaction and magnesium chloride, continue to add the DNA template and primers. Finally, add 0.5 to 2.5 units of DNA polymerase per 50 microliter reaction in a 1.8 milliliter micro fuge tube.
Prepare a master mix solution enough to accommodate for controls as well as pipette transfer loss. Set a micro pipetter to about half the total solution volume and gently mix by pipetting. For each test sample eloquent master mix into PCR tube.
Now for the negative control without template DNA, add all reagents and use sterile water to compensate for the missing DNA volume. Then for the positive control, use a set of template DNA and primers that amplify a known fragment under the same conditions as the experimental PCR samples. PCR thermal cyclers rapidly heat and cool the reaction mixture, allowing for heat induced denaturation of duplex, DNAA kneeling of primers to the plus and minus strands of the DNA template and elongation of the PCR product.
Cycling times are based on the size of the template and the GC content of the DNA for the general formula. Start with an initial denaturation of the template, then initiate 25 to 35 rounds of a three-step temperature cycle program. The first step of these cycles to denature the DNA template.
Then program for optimal, a kneeling of primers about five degrees Celsius below the apparent melting temperature of the primers, followed by the elongation step to bring the polymerase to the DNA template and synthesize the PCR product. Now enter the number of cycles. Next program.
An extended elongation step for completion of synthesis of amplicons. Finally, include a termination step, chilling the mixture to four degrees Celsius. If standard PCR conditions do not yield the desired amplicon, PCR optimization is necessary to attain better results.
So if the reactions didn't work the first time, you want to try to troubleshoot the reaction. And so first, you wanna make sure that the, the problem wasn't human error. And that could happen if you have a pipetting error or if you, if you forgot to add a reagent to the test, one of the test tubes.
Next, you can try altering some of the stringencies of the condition so you can alter the setup of the thermocycler, or you can alter the magnesium concentration is another alternative method. You can also try adding some additives to the reagent to, to troubleshoot the problems. Alternately, consider hot start PCR as a versatile modification in which the initial denaturation time is increased dramatically.
AROS gel electrophoresis is then used to resolve PCR of the GAL three gene from genomic DNA of S seia to determine the optimal magnesium ion concentration for this set of reagents, note, A PCR product of the expected size. 2098 base pairs appears starting at a magnesium concentration of 2.5 millimolar with an optimal concentration at four millimolar on different DNA template amplification of the desired PCR product as a discrete band requires two millimolar magnesium. Reducing the stringency of the reaction to effectively suboptimal amplification conditions produces a smear of non-specific products.
Furthermore, with the overall stringency of the reaction reduction, a lower amount of magnesium ion is required to form an amplicon icon. Thus optimizing PCR while being mindful of the variables can produce the discrete desired product. After watching this video, you should have a good understanding of how to set up and prepare a polymerase chain reaction.
The goal is to understand the variables of each PCR and how they can be manipulated to create the desired amplicon.
