ApaI NEB#R0114

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	DNA products were purified by agarose gel electrophoresis, then digested with ApaI (NEB) at room temperature for 1 h, or with BamHI (NEB) at 37 °C for 1 h, as appropriate. The three digestion products were purified with a DNA Purification Kit (Qiagen) and mixed in equimolar proportions for ligation with T4 ligase (NEB) at 4 °C for 24 h.

Protocol tips
DNA products were purified by agarose gel electrophoresis, then digested with ApaI (NEB) at room temperature for 1 h, or with BamHI (NEB) at 37 °C for 1 h, as appropriate. The three digestion products were purified with a DNA Purification Kit (Qiagen) and mixed in equimolar proportions for ligation with T4 ligase (NEB) at 4 °C for 24 h.

Publication protocol

To disrupt the ivrR recombinase of S. pneumoniae R6 and thereby stabilize the locus in different orientations, the two ~500 bp halves of the recombinase gene were separately amplified using the primer pairs R6hsdSL and Lint, which added an ApaI site, and R6hsdSR and Linr, which added a BamHI site. The ermCB resistance marker was then amplified using template DNA from a macrolide-resistant PMEN1 isolate with primers ermBF and ermBR, which added BamHI and ApaI sites onto the construct, respectively. DNA products were purified by agarose gel electrophoresis, then digested with ApaI (NEB) at room temperature for 1 h, or with BamHI (NEB) at 37 °C for 1 h, as appropriate. The three digestion products were purified with a DNA Purification Kit (Qiagen) and mixed in equimolar proportions for ligation with T4 ligase (NEB) at 4 °C for 24 h. Full-length ligation products were then amplified using primers R6hsdSL and R6hsdSR; this allowed a product around 3 kb in length to be purified through agarose gel electrophoresis. This construct was then reamplified with the same primer pair and used to transform thawed S. pneumoniae R6 cells in the presence of 10 ng of CSP-1 and 5 μl 500 mM calcium chloride. After 2 h incubation, cells were spread on blood agar plates supplemented with 5 mg l−1 erythromycin, and multiple colonies picked for screening using PCRs to identify mutants with different spnIVRhsdS genes, resulting in the isolation of the three patterns found in isolates S. pneumoniae R6 Aa, Ab and Ba.

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