![]() ![]() The PCR performed in a 25 µl reaction mixture containing 25 ng DNA, 1x ThermoPol® buffer (with 2 mM MgCl 2 or MgSO 4), 20-300 nM of primer (for multiplex PCR, primer combinations – maximum 1 µM is total concentration), 0.2 mM dNTPs, 1 U Taq DNA polymerase and (optionally) additional 0.01U Pfu DNA Polymerase (for long products amplification).Ī polymerases mix consisting of 100-500 units of Taq DNA polymerase with 1 unit of Pfu DNA Polymerase greatly increased efficiently of amplification for long bands and the accuracy of the PCR. ![]() The denaturation of genomic DNA is easy with short step at 98☌, 5-10 seconds. This step includes primer binding the target and polymerase extension at once the recommended time for this step is 1 second for each 100 bases of PCR product. PCR steps - the primers binding (usually from 55-60☌) and the polymerase extension (usually from 50☌ to 72☌), we recommend to join into one step as 68-72☌. The optimal annealing temperature for PCR is calculated directly as the value for the primer with the lowest Tm (T m min), our empirical formulae: For primer combinations with very different Tm, the optimal annealing temperature was chosen according to lowest Tm primer (primer with CG content higher then 50% is tolerant to wide annealing Ta, from 55☌ up to 72☌). The range of optimal annealing temperature (Ta) was calculated Tm of primer or optionally plus 6-12☌, and in practice PCR efficiency was tested with gradient annealing temperature using MasterCycler Gradient (Eppendorf). PCR reaction can set up in room temperature and performed without hot-start enzymes. The higher quality of primers is help to save PCR efficiency at changing PCR conditions. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |