Restriction Enzymes AluI

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 inclue: 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 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, 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 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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AluI R6281

Promega

Upstream tips	Protocol tips	Downstream tips
	DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA. DNA digestion was performed on cells in three-dimensional cultures grown for 14 days.Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures. Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period.

Protocol tips
DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA. DNA digestion was performed on cells in three-dimensional cultures grown for 14 days.Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures. Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period.

Protocol tips

DNA digestion was initiated by adding 50 μl of serum-free DMEM containing 0.5 μl of 10 U/μl AluI (Promega, San Luis Obispo, CA) restriction enzyme (5 U per smear) for 30 to 90 minutes and terminated by adding 1 μg/ml ethidium bromide (Fisher Scientific) to stain DNA.
DNA digestion was performed on cells in three-dimensional cultures grown for 14 days.Eight hours of digestion using 60 U of AluI was sufficient for complete digestion of MCF10A-organized acini. The T4-2 aggregates still exhibited partial resistance to digestion after 36 hours of digestion using a total of 600 U of AluI (added 200 U every 12 hours), suggesting that the difference in DNA digestion is not attributable to differences in the amount of DNA contained in the three-dimensional cultures.
Cells in three-dimensional cultures grown for 14 days were first permeabilized for 15 minutes with 0.1% Nonidet P-40, rinsed gently three times in phosphate-buffered saline (PBS), and incubated with serum-free DMEM containing AluI restriction enzyme (Promega) for 24 hours in an incubator at 37°C. Three-dimensional cultures of MCF10A acini, HMT-3522 T4-2 aggregates, and HMT-3522 T4-2 revertants were digested with 20 μl of AluI (100 U/ml media) added twice (total 400 U/reaction) within a 24-hour incubation period.

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest AluI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	2200 bp bands amplified from gDNA of five +B and five 0B mice were removed from the agarose gels, purified with QIAquick Gel Extraction Kit (Qiagen) and then digested with twelve restriction endonucleases (RE): ApaI, HindIII, Bsp120I, MfeI, HpaI, MspI, MboI, AluI, MseI, Ecl136II, HaeIII and SacI (FastDigest, Thermo Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Digested DNA fragments were separated by agarose gel electrophoresis, visualized and photo-documented as above.

Protocol tips
2200 bp bands amplified from gDNA of five +B and five 0B mice were removed from the agarose gels, purified with QIAquick Gel Extraction Kit (Qiagen) and then digested with twelve restriction endonucleases (RE): ApaI, HindIII, Bsp120I, MfeI, HpaI, MspI, MboI, AluI, MseI, Ecl136II, HaeIII and SacI (FastDigest, Thermo Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Digested DNA fragments were separated by agarose gel electrophoresis, visualized and photo-documented as above.

Protocol tips

2200 bp bands amplified from gDNA of five +B and five 0B mice were removed from the agarose gels, purified with QIAquick Gel Extraction Kit (Qiagen) and then digested with twelve restriction endonucleases (RE): ApaI, HindIII, Bsp120I, MfeI, HpaI, MspI, MboI, AluI, MseI, Ecl136II, HaeIII and SacI (FastDigest, Thermo Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Digested DNA fragments were separated by agarose gel electrophoresis, visualized and photo-documented as above.

Manufacturer protocol Publication protocol 1 Relevant paper

AluI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in a previous work [13].

Protocol tips
Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in a previous work [13].

Manufacturer protocol Publication protocol 1 Relevant paper

AluI NEB#R0137

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	After digestion with HaeIII and AluI (NEB, New England Biolabs, USA) DNA was treated with MBN nuclease (NEB) and FastAP alkaline phosphatase (Fermentas, Lithuania).

Protocol tips
After digestion with HaeIII and AluI (NEB, New England Biolabs, USA) DNA was treated with MBN nuclease (NEB) and FastAP alkaline phosphatase (Fermentas, Lithuania).

Publication protocol 1 Relevant paper

AluI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Five microliters of each sample was digested with restriction enzyme AluI (Takara Shuzo).

Protocol tips
Five microliters of each sample was digested with restriction enzyme AluI (Takara Shuzo).

Manufacturer protocol Publication protocol 1 Relevant paper

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