HinfI NEB#R0155

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	RFLP was carried out in 30 μl reaction containing 0.5 μg (10-15 μl) PCR product, RE buffer, 2U of HaeIII / HinfI / XhoI restriction enzyme from NEB [9, 12]. Digestion was carried out for 1 h 30 min. Digests were electrophoresed on 1% agarose gel and band patterns were analyzed by visual comparison.

Protocol tips
RFLP was carried out in 30 μl reaction containing 0.5 μg (10-15 μl) PCR product, RE buffer, 2U of HaeIII / HinfI / XhoI restriction enzyme from NEB [9, 12]. Digestion was carried out for 1 h 30 min. Digests were electrophoresed on 1% agarose gel and band patterns were analyzed by visual comparison.

Publication protocol

vir typing is based on amplification of virulence regulon that encode for different GroupA Streptococcal virulent proteins. Amplification of vir regulon specific PCR amplicon was performed by VUF and SBR primers, as described by Gardiner et al. [12], with few modifications. PCR amplification was carried out in a 50 μl reaction mixture containing 5 μl of template DNA, 1U of TaKaRa LA Taq polymerase, LA PCR Buffer II, 2 mM MgCl2, 200 μM of each of dATP, dGTP, dCTP and dTTP, 1 μl of each 20 μM primers. Cycling conditions were modified to include a final extension at 72 °C for 7 min. Then 5 μl of PCR product was electrophoresed on 0.8% agarose gel to check the quality as well as the quantity of the amplicon. RFLP was carried out in 30 μl reaction containing 0.5 μg (10-15 μl) PCR product, RE buffer, 2U of HaeIII / HinfI / XhoI restriction enzyme from NEB [9, 12]. Digestion was carried out for 1 h 30 min. Digests were electrophoresed on 1% agarose gel and band patterns were analyzed by visual comparison.

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