Restriction Enzymes StyI / EcoT14I / Eco130I

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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StyI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	We performed both single and double digestion. For single digestion we used the 4-base cutter MseI (NEB, R0525S.) For double digestion we used MseI and the 6-base cutter, StyI-HF (NEB, R3500S.) The digestion reaction steps are as following: Normalize the cleaned double stranded cDNA to the appropriate concentration (~30ng/μl.) Dilute MseI (10U/μl) and Styl-HF (20U/μl) enzyme in dilutant A solution (NEB B8001S) to the ratio 1:8 and 1:14, respectively. Set up a 30μl restriction digestion reaction as following: 10μl double-stranded cDNA (a total of 300ng), 3μl Cutsmart buffer (NEB), 1μl diluted MseI, 1μl diluted Styl-HF (substitute with H20 in case of single digestion), and 15μl H2O. In a Thermocycler, incubate the digestion reaction at 37°C for 4 hours, 65°C for 20 min, and hold at 4°C. Proceed immediately to the next step. The sized distribution pattern of ds-cDNA, MseI digested cDNA, Styl digested cDNA, and MseI-Styl double digested cDNA are shown in Fig 4A, 4B, 4C and 4D, respectively.

Protocol tips
We performed both single and double digestion. For single digestion we used the 4-base cutter MseI (NEB, R0525S.) For double digestion we used MseI and the 6-base cutter, StyI-HF (NEB, R3500S.) The digestion reaction steps are as following: Normalize the cleaned double stranded cDNA to the appropriate concentration (~30ng/μl.) Dilute MseI (10U/μl) and Styl-HF (20U/μl) enzyme in dilutant A solution (NEB B8001S) to the ratio 1:8 and 1:14, respectively. Set up a 30μl restriction digestion reaction as following: 10μl double-stranded cDNA (a total of 300ng), 3μl Cutsmart buffer (NEB), 1μl diluted MseI, 1μl diluted Styl-HF (substitute with H20 in case of single digestion), and 15μl H2O. In a Thermocycler, incubate the digestion reaction at 37°C for 4 hours, 65°C for 20 min, and hold at 4°C. Proceed immediately to the next step. The sized distribution pattern of ds-cDNA, MseI digested cDNA, Styl digested cDNA, and MseI-Styl double digested cDNA are shown in Fig 4A, 4B, 4C and 4D, respectively.

Protocol tips

We performed both single and double digestion. For single digestion we used the 4-base cutter MseI (NEB, R0525S.) For double digestion we used MseI and the 6-base cutter, StyI-HF (NEB, R3500S.) The digestion reaction steps are as following:

Normalize the cleaned double stranded cDNA to the appropriate concentration (~30ng/μl.) Dilute MseI (10U/μl) and Styl-HF (20U/μl) enzyme in dilutant A solution (NEB B8001S) to the ratio 1:8 and 1:14, respectively. Set up a 30μl restriction digestion reaction as following:

10μl double-stranded cDNA (a total of 300ng), 3μl Cutsmart buffer (NEB), 1μl diluted MseI, 1μl diluted Styl-HF (substitute with H20 in case of single digestion), and 15μl H2O. In a Thermocycler, incubate the digestion reaction at 37°C for 4 hours, 65°C for 20 min, and hold at 4°C. Proceed immediately to the next step. The sized distribution pattern of ds-cDNA, MseI digested cDNA, Styl digested cDNA, and MseI-Styl double digested cDNA are shown in Fig 4A, 4B, 4C and 4D, respectively.

Publication protocol 1 Relevant paper

EcoT14I (StyI) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	The INHβA PCR products were digested with StyI (Sang et al., 2011) (TaKaRa, Tokyo, Japan). The digestion mixture contained 4 µL PCR products, 1X digestion buffer, and 3.0 U of each enzyme, and was digested overnight at 37°C.	Fragments were separated on 2% agarose gels and were visualized with GelRed staining

Protocol tips
The INHβA PCR products were digested with StyI (Sang et al., 2011) (TaKaRa, Tokyo, Japan). The digestion mixture contained 4 µL PCR products, 1X digestion buffer, and 3.0 U of each enzyme, and was digested overnight at 37°C.
Downstream tips
Fragments were separated on 2% agarose gels and were visualized with GelRed staining

Publication protocol 1 Relevant paper

FastDigest Eco130I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The PCR product was treated at 37°C for 20 min andthen at 65°C for 5 min with the Fast Digest Eco130I (StyI)(Fermentas) enzyme and the expected product sizes weredetermined as 391 and 313 bp, respectively.

Protocol tips
The PCR product was treated at 37°C for 20 min andthen at 65°C for 5 min with the Fast Digest Eco130I (StyI)(Fermentas) enzyme and the expected product sizes weredetermined as 391 and 313 bp, respectively.

Manufacturer protocol Publication protocol 1 Relevant paper

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