Nucleic Acids Research, Vol. 19, No. 8 1953
A method for efficient and rapid cloning of cDNA into Xgtl 0 that facilitates directional sub-cloning into plasmid vectors Nicholas J.P.Ryba, Matthew D.Hall, John B.C.Findlay and Roberto Tirindelli* Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Submitted February 18, 1991 Here we describe a method that reduces handling of valuable cDNA and is easily adaptable to allow cloning into any other vector with a single EcoRI site (Fig. 1). An oligo-nucleotide primer 5' P-dGCGGCCGC(T)17 was synthesised, and was used to replace (dT). as primer for the synthesis of cDNA. This modification incorporates a NotI-site into the cDNA and thus allows directional sub-cloning. The phosphorylation of the primer is critical for the unconventional use of adapters described here. Long non-palindromic SmaI-XmnI-EcoRI adapters (New England Biolabs) were annealed without phosphorylation. A 40-fold excess (over ends) of adapters was ligated with cDNA (4°C, overnight, 6U T4-DNA ligase). The DNA was precipitated with 0.1 volume SM sodium perchlorate, 0.5 volume propan-2-ol at room temperature to remove the majority of the adapters. Because only the cDNA was phosphorylated, only one strand of the adapter was covalently joined to the cDNA. Thus heat-treatment (65°C, 10 min.) generated long non-complementary sticky ends. Residual oligonucleotides were removed by gel-filtration using Sepharose CL4B (Pharmacia) which also achieved a partial sizing of the cDNA as the exclusion limit for Sepharose CL4B is about 550 bp. Similar ligation of the adapters to EcoRI-cut XgtlO, followed by removal of annealed and excess oligonucleotide by heating and 3 sodium perchlorate/propan-2-ol precipitations generated vector with sticky-ends complementary to the adapted cDNA. 5'-phosphorylation prepared the vector to accept such cDNA. Vector and cDNA were annealed by lowering the temperature from 65°C to 16°C over 90 min., and were ligated (overnight, 4°C, 1U T4-DNA ligase). Packaging this DNA (Stratagene, Gigapack plus) produced libraries that contained 5 x 106 independent clones per ug A+RNA. We have generated libraries both from squid (Loligoforbesi) eyes (full-length clones for G-f [1] and for rhodopsin [2] have been isolated from this library) and from rat olfactory epithelium. When plated on BNN93 cells more than 90% of plaques were clear. Minipreparations indicated that > 95% of the plaques formed after plating on BNNJ02 contained recombinant phage with inserts of more than 500 bp, and that the average size of inserts reflected the average size of the cDNA, 700 bp and 800 bp for the squid
*Present address: Istituto Fisiologia Umana, Universita di Parma,
Parma, Italy
and rat libraries respectively. By cutting mini-preparation XDNA with the restriction enzymes, NotI and EcoRI, rapid and directional sub-cloning of cDNA into a suitably prepared plasmid eg. pBluescript (Stratagene) has been achieved.
REFERENCES 1. Ryba,N.J.P., Pottinger,J.D.D., Keen,J.N. and Findlay, J.B.C. (1991) Biochem. J. 273, 225-228. 2. Hall,M.D., Hoon,M.A., Ryba,N.J.P., Keen,J.N., Saibil,H.R. and Findlay,J.B.C. (1991) Biochem. J. 274, 35-40.
Anz,
~~~~EcoRI
\ \
A method for efficient and rapid cloning of cDNA into Xgtl 0 that facilitates directional sub-cloning into plasmid vectors Nicholas J.P.Ryba, Matthew D.Hall, John B.C.Findlay and Roberto Tirindelli* Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Submitted February 18, 1991 Here we describe a method that reduces handling of valuable cDNA and is easily adaptable to allow cloning into any other vector with a single EcoRI site (Fig. 1). An oligo-nucleotide primer 5' P-dGCGGCCGC(T)17 was synthesised, and was used to replace (dT). as primer for the synthesis of cDNA. This modification incorporates a NotI-site into the cDNA and thus allows directional sub-cloning. The phosphorylation of the primer is critical for the unconventional use of adapters described here. Long non-palindromic SmaI-XmnI-EcoRI adapters (New England Biolabs) were annealed without phosphorylation. A 40-fold excess (over ends) of adapters was ligated with cDNA (4°C, overnight, 6U T4-DNA ligase). The DNA was precipitated with 0.1 volume SM sodium perchlorate, 0.5 volume propan-2-ol at room temperature to remove the majority of the adapters. Because only the cDNA was phosphorylated, only one strand of the adapter was covalently joined to the cDNA. Thus heat-treatment (65°C, 10 min.) generated long non-complementary sticky ends. Residual oligonucleotides were removed by gel-filtration using Sepharose CL4B (Pharmacia) which also achieved a partial sizing of the cDNA as the exclusion limit for Sepharose CL4B is about 550 bp. Similar ligation of the adapters to EcoRI-cut XgtlO, followed by removal of annealed and excess oligonucleotide by heating and 3 sodium perchlorate/propan-2-ol precipitations generated vector with sticky-ends complementary to the adapted cDNA. 5'-phosphorylation prepared the vector to accept such cDNA. Vector and cDNA were annealed by lowering the temperature from 65°C to 16°C over 90 min., and were ligated (overnight, 4°C, 1U T4-DNA ligase). Packaging this DNA (Stratagene, Gigapack plus) produced libraries that contained 5 x 106 independent clones per ug A+RNA. We have generated libraries both from squid (Loligoforbesi) eyes (full-length clones for G-f [1] and for rhodopsin [2] have been isolated from this library) and from rat olfactory epithelium. When plated on BNN93 cells more than 90% of plaques were clear. Minipreparations indicated that > 95% of the plaques formed after plating on BNNJ02 contained recombinant phage with inserts of more than 500 bp, and that the average size of inserts reflected the average size of the cDNA, 700 bp and 800 bp for the squid
*Present address: Istituto Fisiologia Umana, Universita di Parma,
Parma, Italy
and rat libraries respectively. By cutting mini-preparation XDNA with the restriction enzymes, NotI and EcoRI, rapid and directional sub-cloning of cDNA into a suitably prepared plasmid eg. pBluescript (Stratagene) has been achieved.
REFERENCES 1. Ryba,N.J.P., Pottinger,J.D.D., Keen,J.N. and Findlay, J.B.C. (1991) Biochem. J. 273, 225-228. 2. Hall,M.D., Hoon,M.A., Ryba,N.J.P., Keen,J.N., Saibil,H.R. and Findlay,J.B.C. (1991) Biochem. J. 274, 35-40.
Anz,
~~~~EcoRI
\ \
