Restriction Enzymes TaaI / HpyCH4III

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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Found 3 matching solutions for this experiment

TaaI (HpyCH4III) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
Because of presence of another restriction site for TaaI (nucleotide 2643–2647, ACT/GT) rather than recognition target site (nucleotide 2710 2714, ACT/GT), straight application of TaaI on the first PCR products was inaccurate. For elimination of first restriction site another semi nested PCR was performed using P5 as a forward and P6 as a reverse primer (Table1). Subsequently amplified fragment (225bp) was digested with TaaI. Briefly the PCR product was first purified using PCR purification kit (MBST,Iran).	fter purification the 10 µl of each purified PCR product was digested with 2 µl TaaI(Fermenta,10U/ µl) in 2 µl 10× buffer Tango and 16 µl nuclease-free water for 16h at 65°C. As enzyme functional control, first PCR product (403bp) was digested with the restriction enzyme. In some cases, the nucleotide sequence of PCR product was determined through Kowsar Company (Iran).

Upstream tips
Because of presence of another restriction site for TaaI (nucleotide 2643–2647, ACT/GT) rather than recognition target site (nucleotide 2710 2714, ACT/GT), straight application of TaaI on the first PCR products was inaccurate. For elimination of first restriction site another semi nested PCR was performed using P5 as a forward and P6 as a reverse primer (Table1). Subsequently amplified fragment (225bp) was digested with TaaI. Briefly the PCR product was first purified using PCR purification kit (MBST,Iran).
Protocol tips
fter purification the 10 µl of each purified PCR product was digested with 2 µl TaaI(Fermenta,10U/ µl) in 2 µl 10× buffer Tango and 16 µl nuclease-free water for 16h at 65°C. As enzyme functional control, first PCR product (403bp) was digested with the restriction enzyme. In some cases, the nucleotide sequence of PCR product was determined through Kowsar Company (Iran).

Manufacturer protocol Publication protocol 1 Relevant paper

HpyCH4III NEB#R0618

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
For some samples, it was necessary to modify the amount of DNA in the PCR, PCR product in the second reaction or PCR product used in the digestion.	Following amplification (verified by agarose gel electrophoresis), PCR products were digested with the restriction enzyme HpyCH4III (New England biolabs, Ipswich, MA, USA). The restriction enzyme profile was identified using NEBcutter33. The digestion was performed in 10 μL containing 0.5 μL of the enzyme (5U/μL), 1 μL of the enzyme’s buffer and 5 μL of PCR product (2–7 μL according to the intensity of PCR products on agarose gels). The digestion was incubated at 37 °C for 3 hours.	All digestion reaction and the equivalent amount of non-digested DNA were visualized in electrophoresis on 3% agarose gel and examined under a UV transilluminator.

Upstream tips
For some samples, it was necessary to modify the amount of DNA in the PCR, PCR product in the second reaction or PCR product used in the digestion.
Protocol tips
Following amplification (verified by agarose gel electrophoresis), PCR products were digested with the restriction enzyme HpyCH4III (New England biolabs, Ipswich, MA, USA). The restriction enzyme profile was identified using NEBcutter33. The digestion was performed in 10 μL containing 0.5 μL of the enzyme (5U/μL), 1 μL of the enzyme’s buffer and 5 μL of PCR product (2–7 μL according to the intensity of PCR products on agarose gels). The digestion was incubated at 37 °C for 3 hours.
Downstream tips
All digestion reaction and the equivalent amount of non-digested DNA were visualized in electrophoresis on 3% agarose gel and examined under a UV transilluminator.

Publication protocol 1 Relevant paper

FastDigest TaaI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The nested PCR product was digested with TaaI and Tru1I FastDigest enzymes (Thermo Fisher Scientific Inc., Waltham, MA, USA) that produce diagnostic band patterns in agarose gel allowing to identify the two targeted species.	With TaaI enzyme, brown trout DNA gives two bands of 205 and 272 bp

Protocol tips
The nested PCR product was digested with TaaI and Tru1I FastDigest enzymes (Thermo Fisher Scientific Inc., Waltham, MA, USA) that produce diagnostic band patterns in agarose gel allowing to identify the two targeted species.
Downstream tips
With TaaI enzyme, brown trout DNA gives two bands of 205 and 272 bp

Manufacturer protocol Publication protocol 1 Relevant paper

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