Stuffer fragment

The current version of the vector is available from C. F. Barbas III at the Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037. This vector contains a number of modifications in the promoter regions designed to minimize recombination. In addition, the vector contains two different stuffer fragments (1200 and 300 bp) in the insertion sites, which is an improvement over using the TT antibody clone as the parent vector. 

The EMBL vector systems have been subjected to many modifications to enhance their usefulness. The most useful modifications involve the addition of more restriction enzyme recognition sites flanking the stuffer fragment and the addition of T3 and T7 RNA polymerase promoters. The T3 and T7 RNA polymerase promoters are used to synthesize 32P-labeled RNAs that can be used as probes for the identification of overlapping A clones. Many of the A vectors described above are available from or have been developed by commercial vendors. As in the case with many basic molecular biology techniques, the components needed to construct a specific recombinant phage library (A vector arms, in vitro packaging extracts, etc.) and even the library can be obtained from commercial sources. 

Analyze the PEG supernatant solution and purified Charon 40 arms by electrophoresis through a 0.7% agarose gel to determine the purity of the Charon 40 arms and the amount of arms remaining in the PEG supernatant. Theoretically, the PEG supernatant solution should contain only the stuffer fragments while the pellet should contain only Charon 40 arms (Fig. 3). If the arms contain a large amount of stuffer fragments the PEG precipitation steps should be repeated. 

Figure 1.2-1. Examples of vectors used for construction of genomic libraries (maximum capacity 24kb). Standard X vectors (XEMBL3, XGEMll, X.DASH, A,EMBL3, and A.SK6) and diphasmid vectors (XSK17 and XSK40) are shown. Sizes are in kilobases and are not shown in the scale. Not all restriction sites are shown. Heavy thick lines represent stuffer fragments open boxes mark plasmid and M13 sequences /acZO is the lac operator sequence. T3, T7, SP6-promoters for T3, T7, and SP6 RNA polymerases.

Oligonucleotides BamHI-Scal connecting vector sequences with the stuffer fragment are physically removed from the vector and stuffer fragments. 

Test of the vector. Before use, the cut vector DNA should be tested for use with appropriate enzymes, the removal of the stuffer fragment and the presence of intact sticky ends at the cloning site for efficient ligation with insert. 

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