The PCR reaction solution (25 μl) consisted of: 0.2 μg of genomic DNA, 0.4 μM of each primer, 1 mM dNTPs, 2 mM MgCl2, 20 mM Tris–HCl, pH 8.8, 50 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100 and 2U Pwo DNA polymerase (Blirt SA DNA-Gdańsk, Poland). 35 cycles were performed, using the Veriti® 96 Well Thermal Cycler (Applied Biosystems, USA), with a temperature profile of 1 min ATM Kinase Inhibitor price at 94°C, 1 min at 60°C and 1 min at 72°C. The amplification products were EPZ-6438 chemical structure analyzed by electrophoresis on 1% agarose

gel stained with ethidium bromide, at a final concentration of 0.5 μg/ml. Specific PCR products were obtained and purified using the ExtractMe Gel-Out Kit (Blirt SA DNA-Gdańsk, Poland). The PCR products were digested with NcoI and BglII or HindIII (NEB, USA), then purified, using the ExtractMe Clean-Up Kit (Blirt SA DNA-Gdańsk, Poland) and ligated into pBAD/myc-HisA plasmid (Invitrogen, USA) selleck chemicals between the NcoI and BglII or NcoI and HindIII sites. The E. coli TOP10 cells were transformed with the ligation mixtures and transformants were examined for the presence of the ssb-like genes, using a gel retardation assay and restriction analysis. One clone was selected and sequenced to confirm the presence of the ssb-like genes. The appropriate pBADDpsSSB, pBADFpsSSB, pBADParSSB, pBADPcrSSB, pBADPinSSB, pBADPprSSB, and pBADPtoSSB recombinant plasmids were

obtained. Table 4 The specific primers for PCR amplification Name Primer sequence fpsssbNcoI 5′ GGA GGA C CA TGG GGA ACG GAA CGT TAA ATA AAG TCA TG 3′ fpsssbHindIII

5′ TTA AAG CTT TTA AAA AGG CAA ATC ATT TTC TAC AG 3′ pcrssbNcoI 5′ TTA CC A TGG GGC GCG GTG TTA ATA AAG TTA TCA TC 3′ pcrssbHindIII 5′ TTA AAG CTT TCA GAA CGG AAT GTC ATC GTC 3′ ptossbNcoI 5′ GGA GGA CC A TGG CAG GAA CAC TCA ATA AAG TTA TGC 3′ ptossbHindIII 5′ TTA AAG CTT TTA AAA GGG TAG ATC ATC TTC CTC 3′ pprssbNcoI 5′ GGA GGA CC A TGG CCA GTC GTG GTG TAA ATA AGG 3′ pprssbBglII 5′ TTA AGA TCT CTA GAA TGG GAT ATC ATC ATC AAA ATC 3′ dpsssbNcoI 5′ TTA CC A TGG GGA TAA ATA AGG CAA TTT TAA TTG GTA ATC TAG 3′ dpsssbHindIII 5′ TTA AAG CTT CTA GAA GGG TAC GTC GTT AC 3′ parssbNcoI 5′ GGA GGA CC A TGG GGC GCG GTG TTA ATA AAG TTA TCA TC 3′ parssbBglII 5′ TTA AGA TCT CTA GAA AGG AAT GTC ATC GTC 3′ pinssbNcoI 5′ TTA Clomifene CC A TGG GGT TTA ACC GAA GCG TAA ACA AAG TAG 3′ pinssbHindIII 5′ TTA AAG CTT CTA AAA AGG AAT ATC ATC ATC GAA ATC 3′ The boldface parts of the primers sequences are complementary to the nucleotide sequences of the ssb-like genes and the underlined parts are the recognition sites for restriction endonucleases. Expression and purification of SSBs The E. coli TOP10 strain transformed with pBADDpsSSB, pBADFpsSSB, pBADParSSB, pBADPcrSSB, pBADPinSSB, pBADPprSSB or pBADPtoSSB was grown at 30°C in Luria-Bertani medium, supplemented with 100 μg/ml of ampicillin, to an OD600 of 0.4, and was induced by incubation in the presence of arabinose, at a final concentration of 0.02%, for 20 h.