DNA has been brought down to a minimum. Indeed, any manipulation performed on DNA, including extraction, digestion, buffer exchange, dephosphorylation, DNA precipitation, column purification, or any other DNA manipulation procedure, is likely to result in some amount of DNA damage and to loss of some of the DNA. With the protocol described here, the plasmids used for assembly are not pre-digested but simply added to the restrictionligation mix. Only one step and one buffer are used, and the time between digestion and ligation is brought to a minimum. No purification step is required between DNA preparation of the input modules and transformation of the library in competent cells. In mitotic cells, the onset of anaphase is triggered by cohesin degradation by separase. Artificial severing of the linkage between sister chromosomes during Afatinib EGFR/HER2 inhibitor metaphase in mitotic cells could result in premature poleward movements of chromosomes , suggesting that the mitotic chromosomes are subject to persistent poleward forces during both metaphase and anaphase. However, in the meiotic oocytes used in this study, there is no DNA replication in the metaphase-arrested eggs and it is unlikely that chromatin cohesion is responsible for restraining poleward movements of DNA beads or sperm chromatin during metaphase. It is more likely that the observed poleward movements of DNA beads or sperm chromatin after anaphase onset are due to direct activation of motors or microtubule depolymerization-based forces as a result of the cell cycle transition. Using undigested plasmids for this procedure rather than digested gel-purified DNA fragments has an added advantage: it allows estimating the relative DNA concentration of the modules more precisely; this is because the relative size difference between modules is much lower for plasmids than for purified inserts. This precision if very important when it comes to ligating many fragments since a module present in too low or too high amount would become a limiting factor and reduce the number of final clones. Unlike for standard cloning, where only one clone is usually required, obtaining the maximum number of independent recombinant plasmids is a necessity for DNA shuffling. A transient breach in vessel wall integrity secondary to growth factordriven active endothelial cell proliferation and sprouting may have resulted in the extravasation of RBCs and the resultant intratumoral hemorrhage. Given the higher free energy of hydrolysis possessed by the pyrophosphate moiety, soon after their initial discovery inositol pyrophosphates were suggested to participate in phosphotransferase reactions. This hypothesis was verified ; recent further work has demonstrated that IP7 phosphorylates its substrates by donating its pyrophosphate b-phosphate moiety to pre-phosphorylated serine residues, generating a novel post-translational modification in the form of pyro-phosphorylated proteins. Two distinct classes of evolutionarily conserved enzymes synthesize inositol pyrophosphates.