Restriction Enzymes PstI

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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PstI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The XPD genotypes (Lys751Gln/A to C) were determined by the PCR amplification and restriction digestion of the products with PstI (Thermo Fisher Scientific Inc. (EU), Lithuania). The PCR amplicon (436 bp) was digested overnight at 37°C with PstI.	The restriction/digestion products were resolved on 3% agarose gel containing 0.5 μg/ml ethidium bromide using a gel electrophoresis system at 100 V for 30–40 min and visualized under UV light. The PstI digestion resulted in two fragments of 290 bp and 146 bp for homozygous wild genotype (Lys/Lys), three fragments of 227 bp, 146 bp, and 63 bp for homozygous variant genotype (Gln/Gln), and four fragments of 290 bp, 227 bp, 146 bp, and 63 bp for heterozygous genotype (Lys/Gln) (Figure 2).

Protocol tips
The XPD genotypes (Lys751Gln/A to C) were determined by the PCR amplification and restriction digestion of the products with PstI (Thermo Fisher Scientific Inc. (EU), Lithuania). The PCR amplicon (436 bp) was digested overnight at 37°C with PstI.
Downstream tips
The restriction/digestion products were resolved on 3% agarose gel containing 0.5 μg/ml ethidium bromide using a gel electrophoresis system at 100 V for 30–40 min and visualized under UV light. The PstI digestion resulted in two fragments of 290 bp and 146 bp for homozygous wild genotype (Lys/Lys), three fragments of 227 bp, 146 bp, and 63 bp for homozygous variant genotype (Gln/Gln), and four fragments of 290 bp, 227 bp, 146 bp, and 63 bp for heterozygous genotype (Lys/Gln) (Figure 2).

Manufacturer protocol Publication protocol 1 Relevant paper

PstI NEB#R0140

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Typically, mtTEC DNA was digested by incubating mtTECs (5 mg of protein) with 5–20 U of appropriate restriction enzyme in a final volume of 30 μL for 30 min at 30°C as follows: 20 U of HhaI (NEB), 5 or 20 U of AvaII (NEB), 20 U of NspI (NEB), 5 U of RcaI (Roche), 5 U of XhoI, 5 U of XhoII, 5 U of PstI (NEB), 20 U of MspI (Roche), and 10 U of SacII (NEB). Identical amounts of restriction enzyme were used in digestion reactions for both circle and hybrid cassette templates.

Protocol tips
Typically, mtTEC DNA was digested by incubating mtTECs (5 mg of protein) with 5–20 U of appropriate restriction enzyme in a final volume of 30 μL for 30 min at 30°C as follows: 20 U of HhaI (NEB), 5 or 20 U of AvaII (NEB), 20 U of NspI (NEB), 5 U of RcaI (Roche), 5 U of XhoI, 5 U of XhoII, 5 U of PstI (NEB), 20 U of MspI (Roche), and 10 U of SacII (NEB). Identical amounts of restriction enzyme were used in digestion reactions for both circle and hybrid cassette templates.

Protocol tips

Typically, mtTEC DNA was digested by incubating mtTECs (5 mg of protein) with 5–20 U of appropriate restriction enzyme in a final volume of 30 μL for 30 min at 30°C as follows: 20 U of HhaI (NEB), 5 or 20 U of AvaII (NEB), 20 U of NspI (NEB), 5 U of RcaI (Roche), 5 U of XhoI, 5 U of XhoII, 5 U of PstI (NEB), 20 U of MspI (Roche), and 10 U of SacII (NEB). Identical amounts of restriction enzyme were used in digestion reactions for both circle and hybrid cassette templates.

Publication protocol 1 Relevant paper

FastDigest PstI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	A total of 250 ng of genomic DNA for each line was double digested with SalI and PstI, PstI and EcoRI, EcoRI and HindIII, or PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA); ligated to adapters (Table 1) using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA); and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments.

Protocol tips
A total of 250 ng of genomic DNA for each line was double digested with SalI and PstI, PstI and EcoRI, EcoRI and HindIII, or PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA); ligated to adapters (Table 1) using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA); and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments.

Protocol tips

A total of 250 ng of genomic DNA for each line was double digested with SalI and PstI, PstI and EcoRI, EcoRI and HindIII, or PstI and MspI (FastDigest restriction enzymes; Thermo Fisher Scientific, Waltham, MA, USA); ligated to adapters (Table 1) using the LigaFast Rapid DNA Ligation System (Promega, Madison, WI, USA); and purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) to eliminate short (<300 bp) DNA fragments.

Manufacturer protocol Publication protocol 1 Relevant paper

PstI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	High-fidelity (HF) ApoI and PstI restriction enzymes were obtained from New England BioLabs Inc. (Ipswich, Massachusetts USA). The optimization of restriction enzyme digestion (Supplementary Fig. 4) was performed on 500 ng of FLO1 cell line genomic DNA and included optimization of enzyme concentration, library purification procedure, PCR cycle optimization and removal of FFPE artefacts.

Protocol tips
High-fidelity (HF) ApoI and PstI restriction enzymes were obtained from New England BioLabs Inc. (Ipswich, Massachusetts USA). The optimization of restriction enzyme digestion (Supplementary Fig. 4) was performed on 500 ng of FLO1 cell line genomic DNA and included optimization of enzyme concentration, library purification procedure, PCR cycle optimization and removal of FFPE artefacts.

Publication protocol 1 Relevant paper

PstI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	After the chromosomal DNA and vector pUC119 (TaKaRa, Kyoto, Japan) were digested by restriction endonucleases PstI (TaKaRa) and EcoRI (TaKaRa), they were ligated with a DNA ligation kit, version 2 (TaKaRa).

Protocol tips
After the chromosomal DNA and vector pUC119 (TaKaRa, Kyoto, Japan) were digested by restriction endonucleases PstI (TaKaRa) and EcoRI (TaKaRa), they were ligated with a DNA ligation kit, version 2 (TaKaRa).

Manufacturer protocol Publication protocol 1 Relevant paper

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