Template dna amount for pcr

If you calculate it, it is: 4,2 microliter. Thank you, Pito. So far I never had this problem - I guess it is because the different volumes and still do not understand how do you get the exact number 4. Ok, let me start from the begging: Start volume - V1 Start concentration - 4. Then, because of the discussion I payed attention that I have different volumes but with the same units, i. If the units were different - easy.

In my logic, the end concentration 0. Then 0. Then if I use the above calculation I get volume 4. The recommended range of MgCl 2 concentration is mM, under the standard reaction conditions specified. In our experiments, at a final dNTP concentration of 0. Usually However, if inhibitors are present in the reaction mix e.

One-half of the total reaction volume is usually sufficient. Amplification parameters depend greatly on the template, primers and amplification apparatus used.

Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. This interval should be extended up to 10 min for GC-rich templates. Usually denaturation for 0. Alternatively, additives facilitating DNA denaturation - glycerol up to vol. 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.