Ligation protocols

Wu, B., Chen, J., Warren, D., Chen, G., Hua, Z., and Danishefsky, S. (2006) Building complex glycopeptides Development of cysteine-free native chemical ligation protocol. Agnew. Chem. Int. Ed. 45, 4116 1125. 

Standard Native Chemical Ligation Protocol General Procedure ... 

Wu B, Chen J, Warren JD, Chen G, Hua Z, Danishefsky SJ (2006) Building complex glycopeptides developmait of a cysteine-fiee native chemical ligation protocol. Angew Chem Int Ed 45 4116 125... 

Protocol 23.1 describes a standard fly miniprep that requires very few flies and produces high-quality DNA. This protocol can also be used to isolate RNA when RNase-ffee conditions are utilized an extra step must be taken to rid the sample of genomic DNA (e.g., RNase-ffee DNase digestion). Genomic DNA prepared as in Protocol 23.1 is then digested with restriction enzymes (Protocol 23.2) and ligated (Protocol 23.3), and used for either plasmid rescue or inverse PCR as described below. 

Key words Expressed protein ligation, Native chemical ligation. Protocol, Protein ligation,... 

The following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

For certain substrates, Fokin, Van der Eycken, and coworkers subsequently discovered that the azidation and ligation steps can be carried out in a one-pot fashion, thereby simplifying the overall protocol (Scheme 6.222) [397]. This procedure eliminates the need to handle organic azides, as they are generated in situ. Other applications of microwave-assisted copper(I)-catalyzed azide-alkyne ligations ( click chemistry ) have been reported [398],... 

Figure 24.1 Flow chart of RACE-PCR protocol adopted with slight modifications from the instruction manual of the Marathon cDNA amplification kit. Poly A+ RNA is used for the generation of an adaptor-ligated cDNA library. Fragments containing the 5 - and the 3 -end of the cDNA coding for the carrier are amplified from the uncloned library with adaptor primers and gene-specific primers. Afterwards, a full-length clone is generated from the individual RACE products by subcloning or end-to-end PCR.

Identify the ligate by specific antibodies as described in Protocol 2.5.4 (Western blot). 

Figure 6. Solid-support-based protocol for the synthesis of a quadrilateral. Beginning with the support containing a closed junction, alternate cycles of restriction and ligation are performed, always at the position indicated as 1 . Selection of the target product (triangle, quadrilateral, pentalateral,...) is determined by the point at which one chooses to restrict at site 2, exposing a sticky end complementary to that exposed by restriction at site 1. This action corresponds to a strand switch (eliminating a zero node), of the sort shown in Figure 8, below. This is emphasized by the lines of different thickness with which the square catenane is drawn.

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