Induced electrocompetent M. smegmatis mc2155:pJV53 cells were prepared as described previously[27] (link). Briefly, after growth to OD600 of ∼0.4 in Middlebrook 7H9 with 0.2% glycerol, 0.05% Tween 80, and 0.2% succinate, cells were induced with 0.2% acetamide, grown for 3 hours, washed three times with ice-cold 10% glycerol, and stored at −80°C. Aliquots (100 µl) were co-electroporated with phage DNA and recombineering substrate, recovered at 37°C in 7H9 containing 10% ADC and 1 mM CaCl2 for ∼2 hours (lysis does not occur until after 3 hours), and plated on 7H10 agar as top agar lawns with approximately 300 µl of M. smegmatis mc2155.
Plaques were picked into 100 µl phage buffer (10 mM Tris-HCl, pH 7.5; 10 mM MgSO4; 68.5 mM NaCl; 1 mM CaCl2). One microliter was PCR amplified with flanking primers (25–35 bp) annealing upstream and downstream of the mutant allele, or by Deletion Amplification Detection Assay (DADA)-PCR using Platinum Taq High Fidelity DNA Polymerase (Invitrogen) and an upstream primer whose 3′ end anneals over the deletion junction. DADA-PCR parameters were similar to those described for MAMA-PCR[30] (link), with the combined annealing and extension step performed at or just above the melting temperature of the DADA-PCR primer. Plaques containing mixtures of deletion and wild-type DNA were picked into 100 µl buffer, and 10 µl of 10−3, 10−4 and 10−5 dilutions were plated with 300 µl M. smegmatis cells. Either individual plaques from the 10−4 and 10−5 plates or lysates from 10−3 or 10−4 plates were screened for the presence of the mutation by PCR as described above.
Plaques were picked into 100 µl phage buffer (10 mM Tris-HCl, pH 7.5; 10 mM MgSO4; 68.5 mM NaCl; 1 mM CaCl2). One microliter was PCR amplified with flanking primers (25–35 bp) annealing upstream and downstream of the mutant allele, or by Deletion Amplification Detection Assay (DADA)-PCR using Platinum Taq High Fidelity DNA Polymerase (Invitrogen) and an upstream primer whose 3′ end anneals over the deletion junction. DADA-PCR parameters were similar to those described for MAMA-PCR[30] (link), with the combined annealing and extension step performed at or just above the melting temperature of the DADA-PCR primer. Plaques containing mixtures of deletion and wild-type DNA were picked into 100 µl buffer, and 10 µl of 10−3, 10−4 and 10−5 dilutions were plated with 300 µl M. smegmatis cells. Either individual plaques from the 10−4 and 10−5 plates or lysates from 10−3 or 10−4 plates were screened for the presence of the mutation by PCR as described above.
Free full text: Click here
