![]() The resulting PCR products were sequenced using the original PCR and. The GC contents are 55.8 and 45 respectively. All primers were tested for dimer formation using AmplifX 1.7.0 (by Nicolas Jullien. I tried using Pfu Hifidelity and Kapa Hifi. New technologies are emerging that are enabling the cloning of larger pieces of DNA. Here are some hints on how to get the most from those kinds of reactions.įirst of all, start with a DNA polymerase that can amplify large fragments of DNA. Our Q5® DNA polymerase can successfully amplify 10 kb amplicons from complex genomic samples and 20 kb amplicons from simpler genomic templates. The etiological agent of infectious ovine epididymitis is Brucella ovis and for its direct indication in clinical samples several PCR protocols are proposed. For larger sizes than that, our LongAmp ® Taq DNA polymerase, a mixture of Taq DNA Polymerase and a proofreading DNA Polymerase is capable of successfully amplifying up to 30 kb even from complex genomic templates. AmplifX: Manage, test and design your primers for PCR. And so for your denaturation and extension steps, only use as high a temperature and for as long as is required by your experiment. Automatic calculation of quality score (TM Santa-Lucia method, length, GC, autodimer, complexity. Selected primers (Table 1) were finally checked via in silico PCR using AmplifX (version 1.7.0 Jullien, 2013). Note that our LongAmp Taq DNA Polymerase actually uses a 65 degree extension temperature. Vortexing and excessive pipetting can damage your DNA, even fragment it. For 20 kb targets and above, avoid pipetting once you've added your DNA to your reaction.Īnd finally, avoid freeze/thaw cycles of your DNA. Your DNA stocks can be aliquoted and frozen at minus 20 degrees C while your working stocks at lower concentrations can be kept at four degrees C.
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