Standard Polymerase Chain Reaction (PCR) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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< < | PCR reactions produce an amplified product of a template DNA. In addition to the template the reactions include forward and reverse primers, buffer, dNTPs, DNA polymerase and water. When more than a few reactions are being made (5+), it is advisable to use a mastermix. A mastermix is a batch of all of the ingredients common to all the reactions, i.e., water, buffer, polymerase, dNTPs. To make a mastermix, take the amount of each of the individual ingredients needed (ex: 3 μl 10x dNTPs) and multiply it by (number of reactions + 1). Remember to include controls in the total reaction count. So if 8 total reactions are being run, the mastermix would contain 27 μl 10x dNTPs. When many PCR reactions are being run (15+) it often becomes necessary to add more than just 1 extra batch for the final volume. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
> > | PCR reactions produce an amplified double-stranded DNA product from template DNA. In addition to the template, the reactions include forward and reverse primers, buffer, dNTPs, DNA polymerase and water. When mixing together a PCR reaction or a mastermix (see below), the order does matter. Be sure to add water and buffer first and then NTPs and polymerase last. Polymerases are enzymes and can be destroyed if diluted into a solution lacking the proper buffer. For some reactions, it can also be important to add polymerase only directly before thermocycling or on ice to avoid any activity before the PCR program begins. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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< < | Mastermixes not only save time and materials, but they also allow for much greater precision. Measuring out 0.3 μl of 100x Taq polymerase with a pipette is very inaccurate, however, 2.7 μl of the same Taq can go into the MM, from which volumes of higher precision can be withdrawn. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
> > | A basic PCR protocol consists of cycles of three steps: | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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< < | A basic PCR protocol consists of three steps: | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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< < | The exact temperature and timing of each step may vary depending on the polymerase being used (such as Taq or Phusion). PCR protocols can also vary depending on the template: extracted genomic DNA is typical but PCRs can also be performed on DNA obtained from bacterial liquid cultures or colonies from plates. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
> > | The exact temperature and timing of each step may vary depending on the polymerase being used (such as Taq or Phusion). PCR protocols can also vary depending on the template: purified plasmid or genomic DNA is typical but PCRs can also be performed on DNA released directly from bacterial liquid cultures or colonies from plates. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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> > | Tip: Use a Mastermix: When more than a few reactions are being made (5+), it is advisable to use a mastermix. A mastermix is a premixed batch of all of the ingredients common to all of the reactions (e.g., water, buffer, polymerase, dNTPs). To make a mastermix, take the amount of each of the individual ingredients needed (ex: 3 μl 10× dNTPs) and multiply it by (number of reactions + 1). Remember to include controls in the total reaction count. So if 8 total reactions are being run, the mastermix would contain 27 μl 10× dNTPs. When many PCR reactions are being run (15+) it often becomes necessary to add more than just 1 extra batch for the final volume. In this case, you can add an extra 10% of each component. Mastermixes not only save time, but they also allow for much greater precision in mixing the right amounts of PCR components. Measuring out 0.3 μl of 100× Taq polymerase with a pipette is very inaccurate, however, 2.7 μl of the same Taq can go into the mastermix, a volume that can be pipetted with much higher precision. When making a mastermix be sure to add enzyme last so that it only experiences proper buffer conditions. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Basic Conditions using standard Taq polymeraseA typical reaction has a final volume of 30 μl, a template concentration of 0.1ng/μl, and primer concentrations of 500nM each. This chart shows the volumes of various ingredients that should be used. These PCR conditions are suitable for products ranging up to 3 kb in length. PCRs that result in longer products may require optimization of the dNTP and primer concentrations and the use of special DNA polymerase kits.
Basic conditions using NEB Phusion HF PolymeraseWhen using Phusion polymerase, use the NEB calculator to approximate your annealing temperatures. For a 20 ul reaction
Agarose Gel ElectrophoresisIn order to analyze PCR results, the products are run on an agarose gel and the resulting gel is observed in UV light. First, the gel has to be made. A standard 1% agarose gel uses 1g of agarose for every 100 ml of buffer. A different percentage may be used, and gels with less than 1% agarose may be used to clearly distinguish products of very similar sizes. For a standard 50 ml gel, add .5g agarose and 50 ml TAE (1X) buffer to a 125ml flask and heat for 1:30 minutes. Meanwhile, assemble a gel rig and find a comb with an appropriate number of wells, then place the comb into the rig. After heating add 2.5 μl SYBR Safe (5μl SYBR Safe for every 100mL gel) and swirl to mix. Pour the liquid from the flask into the rig and wait about 30 minutes for it to solidify. Once the gel has solidified, gather all PCR products that are to be run, an appropriately sized ladder, and 6x loading dye. The latter two may be found in the 4蚓 fridge in the computer room adjacent to the gel area. First, load 6-7 μl of ladder into the first well. The easiest way to combine dye and DNA is to cut out a 4x4 sheet of parafilm, make a drop of 1 μl of dye onto the parafilm for each sample to be run. Next, add 5 μl of PCR product to the dye and pipette up and down to homogenize. Once all samples are combined with dye, load them into the gel, making note of what sample goes into what lane.Whole-Cell PCR
Colony PCR
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