Restriction Enzymes BsmFI / FaqI

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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Found 2 matching solutions for this experiment

FaqI (BsmFI) (2 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Genomic DNA was extracted as a template from each embryo following the HotSHOT protocol as described by Meeker et al.4 when the desired assays were done. alas1smu350/smu350 and alas1Δ2/Δ2 mutants were genotyped by PCR followed by FaqI (Thermo Scientific, San Jose, CA, USA) and BsrI (New England Biolabs, Ipswich, MA, USA) digestion, respectively. The alas1smu350/smu350 PCR products were digested by FaqI into two fragments of 57 bp and 85 bp, the alas1Δ2/Δ2 PCR products were digested by BsrI into two fragments of 43 bp and 97 bp, whereas the WT PCR products were resistant to the digestion.

Protocol tips
Genomic DNA was extracted as a template from each embryo following the HotSHOT protocol as described by Meeker et al.4 when the desired assays were done. alas1smu350/smu350 and alas1Δ2/Δ2 mutants were genotyped by PCR followed by FaqI (Thermo Scientific, San Jose, CA, USA) and BsrI (New England Biolabs, Ipswich, MA, USA) digestion, respectively. The alas1smu350/smu350 PCR products were digested by FaqI into two fragments of 57 bp and 85 bp, the alas1Δ2/Δ2 PCR products were digested by BsrI into two fragments of 43 bp and 97 bp, whereas the WT PCR products were resistant to the digestion.

Protocol tips

Genomic DNA was extracted as a template from each embryo following the HotSHOT protocol as described by Meeker et al.4 when the desired
assays were done. alas1smu350/smu350 and alas1Δ2/Δ2 mutants were genotyped by PCR followed by FaqI (Thermo Scientific, San Jose, CA, USA) and BsrI (New England Biolabs, Ipswich, MA, USA) digestion, respectively.
The alas1smu350/smu350 PCR products were digested by FaqI into two fragments of 57 bp and 85 bp, the alas1Δ2/Δ2 PCR products were digested
by BsrI into two fragments of 43 bp and 97 bp, whereas the WT PCR products were resistant to the digestion.

Manufacturer protocol Publication protocol 1 Relevant paper

BsmFI NEB#R0572

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The precipitated amplification products were digested with the tagging enzyme BsmFI in a 50-μl reaction mixture containing 0.2 mM each dNTP, 1× NEB Buffer 4, 1× bovine serum albumin (BSA), and 2 U of BsmFI (NEB) for 1 h at 65°C. Restriction products were filled in by the addition of 3 U of T4 polymerase (NEB) to the reaction mixture and incubated for 20 min at 12°C. The reaction mixture was diluted to a volume of 200 μl with LoTE, and the products were extracted with PC8 and precipitated as described above.

Protocol tips
The precipitated amplification products were digested with the tagging enzyme BsmFI in a 50-μl reaction mixture containing 0.2 mM each dNTP, 1× NEB Buffer 4, 1× bovine serum albumin (BSA), and 2 U of BsmFI (NEB) for 1 h at 65°C. Restriction products were filled in by the addition of 3 U of T4 polymerase (NEB) to the reaction mixture and incubated for 20 min at 12°C. The reaction mixture was diluted to a volume of 200 μl with LoTE, and the products were extracted with PC8 and precipitated as described above.

Protocol tips

The precipitated amplification products were digested with the tagging enzyme BsmFI in a 50-μl reaction mixture containing 0.2 mM each dNTP, 1× NEB Buffer 4, 1× bovine serum albumin (BSA), and 2 U of BsmFI (NEB) for 1 h at 65°C. Restriction products were filled in by the addition of 3 U of T4 polymerase (NEB) to the reaction mixture and incubated for 20 min at 12°C. The reaction mixture was diluted to a volume of 200 μl with LoTE, and the products were extracted with PC8 and precipitated as described above.

Publication protocol 1 Relevant paper

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