Issue link: http://life-technologies.uberflip.com/i/330570
We successfully constructed our vectors using the standard One Shot ® TOP10 protocols. A low cloning efficiency is expected, partly since the vector is large but also because there's only one sticky end made by the AvrII/BstZ17I digest. In addition, the ratio of insert to vector needs to be controlled, as well as the total amount of DNA in the transformation. Using homemade competent cells may not provide high enough cloning efficiency for this type of ligation. For cloning a two-chain gene, we recommend using the following strategy: 1. Generate cDNA fragment for first chain (heavy chain of IgG, for example) with AvrII/blunt overhang restriction sites by PCR by designing restriction sites at the 5' ends of your PCR primers (Figure 1). Note: Cloning using the vector's BstZ17I site alone gave us a much lower number of positive colonies than with the double-digest strategy. 2. Cut pCHO 1.0 with AvrII and BstZ17I; cut PCR product with AvrII and your engineered blunt-cutter. a. We tested both a simultaneous AvrII/Bstz17I digest of the plasmid or sequential (Bstz17I then AvrII) digest and obtained similar results. Because overdigestion with BstZ17I is more difficult (since the enzyme does not withstand extended incubation at 37°C), the following approach is justified: i. Digest with BstZ17I. ii. Confirm vector linearization on a gel. iii. Digest with AvrII overnight. iv. Either gel- or column-purify the digest to remove the restriction enzymes. Vector Construction and Preparation Tip: Tip: 10 Life Technologies ™ | Vector Construction and Preparation