---+ Standard Polymerase Chain Reaction (PCR) 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. A basic PCR protocol consists of cycles of three steps: * 1) Denaturation: separating double-stranded DNA strands by heating * 2) Annealing: binding primers to complementary DNA sequences on the template * 3) Extension: addition of nucleotides to the primers making copies of the target DNA sequence, catalyzed by the polymerase The exact temperature and timing of each step may vary depending on the polymerase being used (such as <a href="#taq">Taq</a> or <a href="#phusion">Phusion</a>). 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 <a href="#culture_PCR">bacterial liquid cultures</a> or <a href="#colony_PCR">colonies from plates</a>. *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. <a id="taq"></a> ---++ Basic Conditions using standard Taq polymerase A 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. %TABLE{dataalign="center"}% | *Template* | *10µM UF primer* | *10µM DR primer* | *10x buffer* | *2mM dNTPs* | *5U/µL taq* | *ddH<sub>2</sub>O* | *Total* | | X | 1.5 | 1.5 | 3 | 3 | 0.3 | 20.7 - X | 30 μl | 10x dNTPs are 2 mM in _each_ dNTP. If the stock concentration of DNA is 1ng/μl, 3 μl would give the desired concentration of template. 1.5 μl of a 10μM primer gives a final concentration of 500nM. Water is added as needed to create a final volume of 30 μl. In the thermocycler, make sure that the program "PCR" contains the appropriate temperatures and times. The most important step to check, is that the 72°C elongation step should be roughly 1 minute for every 1000bp of the longest PCR product. If the longest expected product is 2500bp, then 2.5 minutes at 72°C should be appropriate. For most PCRs, 30-40 cycles should be appropriate. *Further Reading* * [[http://www.neb.com/nebecomm/products/protocol54.asp][New England Biolabs Taq PCR Optimization Page]] <a id="phusion"></a> ---++ Basic conditions using NEB Phusion HF Polymerase When using Phusion polymerase, use the [[http://tmcalculator.neb.com/#!/][NEB calculator]] to approximate your annealing temperatures. For a 20 ul reaction %TABLE{dataalign="center"}% | *Template* | *10µM UF primer* | *10µM DR primer* | *5x Phusion buffer* | *10mM dNTPs* | *Phusion poly* | *ddH<sub>2</sub>O* | *Total* | | X | 1.0 | 1.0 | 4 | 0.4 | 0.2 | up to 20ul | 20 μl | For a 50 ul reaction %TABLE{dataalign="center"}% | *Template* | *10µM UF primer* | *10µM DR primer* | *5x Phusion buffer* | *10mM dNTPs* | *Phusion poly* | *ddH<sub>2</sub>O* | *Total* | | X | 2.5 | 2.5 | 10 | 1 | 0.5 | up to 50µl | 50 μl | Final Concentrations %TABLE{dataalign="center"}% | *Template* | *For primer* | *Rev primer* | *5x Phusion buffer* | *10mM dNTPs* | *Phusion poly* | *ddH<sub>2</sub>O* | | <250 ng | 0.5µM | 0.5µM | 1x | 200µM | 1.0 units/50ul PCR | - | NEB recommends a template amount (for 50µl reaction) of 50 ng - 250 ng for genomic DNA and 1 pg - 10 ng for plasmid or viral DNA. Although the recommended Phusion polymerase concentration is 1.0 units/50 ul rxn (20 units/ml) this concentration may have to be changed based on amplicon length and difficulty. The concentration may vary from (0.5-2 units/50 ul rxn) but should not exceed 2 units/50 ul rxn, especially for amplicons longer than 5 kb. Thermal cycler conditions: * Denaturation: An initial denaturation of 30s at 98°C is sufficient for most amplicons from pure DNA templates; however, longer denaturation times can be used (up to 3 m) for templates that may require it. During thermocycling, this step should be kept to a minimum; typically, NEB recommends a 5-10 s denaturation at 98°C for most templates. * Annealing: Note that the annealing temperatures required for use with Phusion tend to be higher than with other polymerases. Use the [[https://tmcalculator.neb.com/#!/main][NEB calculator]] to approximate your annealing temperatures. Typically, primers greater than 20 nucleotides in length anneal for 10-30 seconds at 3°C above the Tm of the lower Tm primer. * Extension: Times are dependent on amplicon length and complexity. Generally, an extension time of 15 seconds per kb can be used. For complex amplicons, such as genomic DNA, an extension time of 30 seconds per kb is recommended. Notes: * The PCR products generated using Phusion polymerase have blunt ends. * Phusion PCR kits frequently include DMSO, which is an additive that can improve the yield of some PCR reactions, particular those with high GC content or templates with secondary structures such as hairpin loops. DMSO is typically added at a final concentration of ~3% (aka 1.5 ul of DMSO per 50 ul reaction). If a high concentration of DMSO is needed (~10%) the annealing temperature of the reaction should be decreased by ~6°C as described [[https://www.neb.com/protocols/2012/06/01/guidelines-for-pcr-optimization-with-phusion-high-fidelity-dna-polymerase][here]]. ---++ Agarose Gel Electrophoresis In 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°C 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. <a id="culture_PCR"></a> ---++ Whole-Cell PCR * Grow fresh overnight cultures of the cells to be used as template in LB. * *Important:* Do NOT use _E. coli_ grown in Davis Minimal (DM) media as template for whole-cell PCR. Ingredients in DM interfere with reliable amplification. (Probably the high concentration of citrate added as a chelating agent). * Amplification from frozen cultures is also not as reliable as from freshly grown cells. * Dilute cells 1000x fold into ddH<sub>2</sub>O (NOT saline) in a 1.7 ml Eppendorf tube or 96-well microplate. * Too many cells or too much residual media is a common source of failure for whole-cell PCR. * Add the diluted cell mixture as 1/10th the final volume of the PCR reaction. * Add an initial denaturing step of 10 minutes to your PCR program at 94°C to lyse the cells. <a id="colony_PCR"></a> ---++ Colony PCR * A colony from an overnight plate can be used as PCR template. Caveat: Due varying sizes of colonies, the difficulty of picking-up all cells from a colony and other factors, expect this protocol to work 80% of the time. * <strong>Important: </strong>Lab made phusion polymerase is NOT recommended for this protocol (it will greatly lower the chance of your PCR working) * Colonies to be used should have grown to a visible easy-to-pick size * Ideally, colonies are well spread in the plate, such to avoid cross-contamination with other colonies * Set a 1.7 ml Eppendorf tube (or 96-well microplate) with 250µL dH2O. * Have your PCR mix ready with all components - except cells - set on ice. * Scrape-off a whole colony from the plate - you can use a sterile pippette tip for this * Transfer the picked colony (all of it) into the water in the Eppendorf tube by scrapping off the cells from the pippette tip by rubbing it against the wall of the tube several times. * Vortex well the re-suspended colony (~10 seconds). * For a standard 50µL PCR reaction: add 2µL (immediately after vortexing) of the diluted colony mixture to 48µL of PCR mix in a PCR tube. * Add an initial denaturing step of 10 minutes to your PCR program at 98°C to lyse the cells.
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