Restriction Enzymes Hin1II / NlaIII

A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence. The four most common types of restriction enzymes include: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). The most common challenges with restriction digest include- 1. inactivation of the enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, the optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with the activity of the other. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.

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Hin1II (NlaIII) (5 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Following DNA amplification, double digestion of PCR products was performed according to the manufacturer's instructions using the AluI enzyme recognising AG∧CT sites (Thermo Fisher Scientific, Waltham, Massachusetts, USA, catalogue no. ER0011) and the Hin1II (NlaIII) enzyme recognising CATG∧ sites (Thermo Fisher Scientific, catalogue no. ER1831). Briefly, 10 μl of the PCR reaction mixture was mixed with 18 μl of Molecular Biology Reagent Water (Sigma-Aldrich, Saint Louis, Missouri, USA), 2 μl of 10X Buffer Tango, 2 μl of AluI and 4 μl of Hin1II. The mixture was spun down for 30 s and incubated at 37 °C overnight.

Protocol tips
Following DNA amplification, double digestion of PCR products was performed according to the manufacturer's instructions using the AluI enzyme recognising AG∧CT sites (Thermo Fisher Scientific, Waltham, Massachusetts, USA, catalogue no. ER0011) and the Hin1II (NlaIII) enzyme recognising CATG∧ sites (Thermo Fisher Scientific, catalogue no. ER1831). Briefly, 10 μl of the PCR reaction mixture was mixed with 18 μl of Molecular Biology Reagent Water (Sigma-Aldrich, Saint Louis, Missouri, USA), 2 μl of 10X Buffer Tango, 2 μl of AluI and 4 μl of Hin1II. The mixture was spun down for 30 s and incubated at 37 °C overnight.

Protocol tips

Following DNA amplification, double digestion of PCR products was performed according to the manufacturer's instructions using the AluI enzyme recognising AG∧CT sites (Thermo Fisher Scientific, Waltham, Massachusetts, USA, catalogue no. ER0011) and the Hin1II (NlaIII) enzyme recognising CATG∧ sites (Thermo Fisher Scientific, catalogue no. ER1831). Briefly, 10 μl of the PCR reaction mixture was mixed with 18 μl of Molecular Biology Reagent Water (Sigma-Aldrich, Saint Louis, Missouri, USA), 2 μl of 10X Buffer Tango, 2 μl of AluI and 4 μl of Hin1II. The mixture was spun down for 30 s and incubated at 37 °C overnight.

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest Hin1II

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	A volume of 10 μl of PCR products was digested for 1 h at 37°C with XagI (FastDigest, Fermentas) for IL-17A G197A and NlaIII (FastDigest, Fermentas) for IL-17F A7488G. The digested PCR products were electrophoresed on 5% (w/v) agarose gel prestained with ethidium bromide at 70 V for 50 min and visualized using the FluorChem 5500 imaging system (Alpha Innotech Corp., San Leandro, CA, USA).

Protocol tips
A volume of 10 μl of PCR products was digested for 1 h at 37°C with XagI (FastDigest, Fermentas) for IL-17A G197A and NlaIII (FastDigest, Fermentas) for IL-17F A7488G. The digested PCR products were electrophoresed on 5% (w/v) agarose gel prestained with ethidium bromide at 70 V for 50 min and visualized using the FluorChem 5500 imaging system (Alpha Innotech Corp., San Leandro, CA, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

NlaIII NEB#R0125

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Digest 164 bp products of PCR was completed at the condition of 5 units NlaIII (New England Biolabs) at 37°C for 4 hours. Restriction fragments were analyzed using 3% ethidium bromide-stained agarose gel.

Protocol tips
Digest 164 bp products of PCR was completed at the condition of 5 units NlaIII (New England Biolabs) at 37°C for 4 hours. Restriction fragments were analyzed using 3% ethidium bromide-stained agarose gel.

Publication protocol 1 Relevant paper

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