How much dna should i use for pcr

If, on the other hand, the amount of template is too high, the probability of primers annealing to other not one hundred percent complimentary sequences, as well as the formation of primer dimers, will increase, which will result in the amplification of by-products. Following purification, it is necessary to determine the concentration of the DNA to be able to define the volume that is required for the PCR setup. While agarose gel electrophoresis may serve to provide an estimate, this method is far from accurate.

UV-Vis spectrophotometry has been established as the gold standard for the quantification of nucleic acids; this direct and therefore easy and quick method measures absorbance of the sample at nm, and concentration is calculated with the help of a conversion factor.

RNA, protein, salts , this method will reach its limitations. In the case of very low concentrations, the readings will soon become too inaccurate to be of use, and contaminations will lead to sometimes enormous overestimation of the actual value. In this case, quantification using fluorescence may present an alternative. This technique is based on the use of a fluorescent dye e.

DNA template concentration. The concentration of DNA template depends on the source. Concentration of dNTPs. DNA polymerase. Different polymerases are commercially available and are summarized in DNA polymerases. Polymerase buffer. All DNA polymerases are supplied with their own optimal polymerase buffer. The standard polymerase buffer works well for a wide range of templates and primers but may not be optimal for any particular combination.

High concentrations of chelating agents such as EDTA and negatively charged ionic groups such as phosphates should be avoided. GC content of DNA template. 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. 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. Once all samples are combined with dye, load them into the gel, making note of what sample goes into what lane. 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.

Too many cells or too much residual media is a common source of failure for whole-cell PCR. Important: 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.

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.