Strain and media
Escherichia coli XL10-Gold and DH5α were used for routine DNA manipulations. Bacterial cells were cultured in LB medium (0.5% yeast extract, 1% peptone and 1% NaCl) supplied with 100 μg/ml of ampicillin, 0.1 mM IPTG and 0.004% X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) when necessary. Genomic DNA of Saccharomyces cerevisiae S288c (MATα SUC2 mal gal2 mel flo1 flo8-1 hap1 ho bio1 bio6) [Mortimer & Johnston 1986 was used as template for amplification of the LEU2 gene.
Construction of T-vector
The stuffer DNA used in this work was derived from a fragment of the S. cerevisiae URA 3 gene present in plasmid pNKY51 [Alani et al. 1987 and was obtained by PCR using the following primers: PXCM-1 (5′-AAGGTACCGATATCTCCAATACTTGTATGGAGGGCACAGTTAAGCC-3′) and PXCM2 (5′-AAGAGCTCGATATCCTCCAATACTCCTTTGGATCCCTTCCCTTTGCAAATAGT-3′). Primer PXCM-1 contains restriction sites for Sac I, Eco RV and Xcm I while PXCM-2 has sites for Kpn I, Eco RV and Xcm I (all sites are underlined). Both primers have sequences complementary to URA3 which allow amplification of a ~600 pb stuffer DNA fragment. PCR was carried out in a volume of 50 μL containing 1.5 ng pNKY51, 0.2 mM dNTP, 0.2 μM each primer, 1× PCR buffer (100 mM Tris–HCl [pH 8.5], 500 mM KCl), 2 mM MgCl2 and 2 U Taq polymerase (LCG Biotechnology). Amplification was performed for 30 cycles of 94°C/45 s, 65°C/45 s, 72°C/40 s after an initial denaturation step of 94°C/45 s. A final extension step was performed for 2 min/72°C. The resulting amplicon was purified with UltraClean PCR Clean-Up Kit (MO BIO) and digested with Sac I and Kpn I following ligation to pBlueScript® II KS digested with the same enzymes.
Cloning efficiency
To test cloning efficiency of both vectors, the S. cerevisiae LEU2 gene was cloned after amplification from yeast genomic DNA using Taq polymerase (Invitrogen) or Phusion (Finnzymes) and primers 5-leud (5′-GAGATCTATATATATTTCAAGGATATACCATTCTAATG-3′) and 3-leud (5′-GAGATCTGTTTCATGATTTTCTGTTACACC-3′). Both amplification reactions were carried out in a volume of 50 μL. For amplification with Taq polymerase, 10 ng genomic DNA was added to a reaction which included 1× PCR buffer (200 mM Tris–HCl [pH 8.4], 500 mM KCl), 2 mM MgCl2, 0.2 mM dNTP mixture, 0.2 μM each primer and 2 U Taq polymerase. The reaction was performed for 30 cycles of 94°C/45 s, 55°C/30 s, 72°C/1.5 min after an initial denaturation of 94°C/45 s. The final extension was accomplished for 10 min/72°C. The PCR system with Phusion was carried out with 10 ng genomic DNA, 1× Phusion HF buffer (1.5 mM MgCl2), 0.2 mM dNTP, 0.5 μM each primer and 0.5 U Phusion DNA polymerase. The PCR program was: 30 s at 98°C for initial denaturation following 30 cycles of 98°C/10 s, 61°C/30 s, 72°C/30 s with a final extension of 72°C/5 min. PCR products were purified as described previously and ligated into the constructed cloning vectors. Ligation was carried out in a final volume of 10 μL with a vector:insert ratio of 1:5. The system included 1 U of T4 DNA ligase (USB) and 1× reaction buffer (66 mM Tris–HCl [pH 7.6], 6.6 mM MgCl2, 10 mM DTT, 66 μM ATP), and incubation was carried out at 16°C for 16 h following transformation of E. coli DH5α cells.