Restriction Enzymes BciVI / BfuI

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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BciVI NEB#R0596

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Four out of the five selected Type IIS REs were tested (Fig. 4b). The RE-digestion and ligation solution contained 1 µL of Type IIS RE (MboII [R0148S], HphI [R0158S], BmrI [R0600S] or BciVI [R0596S] NEB), 0.3 µL of 1.25 μM L(G/A)-Boligo, 0.3 µL of 1.25 μM R(G/A)-Boligo, 3 µL of fragment (the concentration of fragment is recommended in section “Barcoding”), and 4.6 µL of Blunt/TA Ligase Master Mix. The RE-digestion mixture was incubated at 37 °C for 1 h, and two microliters of the solution was used as template in PCR to amplify barcoded fragment by using corresponding PS-modified Aoligos. PCR product was purified and treated by using iodine solution as specified in section “PCR” and “Iodine-based chemical reaction”. All the non-modified oligos used to amplified the fragments with the 1-nt SEs are listed in Supplementary Data 1.

Protocol tips
Four out of the five selected Type IIS REs were tested (Fig. 4b). The RE-digestion and ligation solution contained 1 µL of Type IIS RE (MboII [R0148S], HphI [R0158S], BmrI [R0600S] or BciVI [R0596S] NEB), 0.3 µL of 1.25 μM L(G/A)-Boligo, 0.3 µL of 1.25 μM R(G/A)-Boligo, 3 µL of fragment (the concentration of fragment is recommended in section “Barcoding”), and 4.6 µL of Blunt/TA Ligase Master Mix. The RE-digestion mixture was incubated at 37 °C for 1 h, and two microliters of the solution was used as template in PCR to amplify barcoded fragment by using corresponding PS-modified Aoligos. PCR product was purified and treated by using iodine solution as specified in section “PCR” and “Iodine-based chemical reaction”. All the non-modified oligos used to amplified the fragments with the 1-nt SEs are listed in Supplementary Data 1.

Protocol tips

Four out of the five selected Type IIS REs were tested (Fig. 4b). The RE-digestion and ligation solution contained 1 µL of Type IIS RE (MboII [R0148S], HphI [R0158S], BmrI [R0600S] or BciVI [R0596S] NEB), 0.3 µL of 1.25 μM L(G/A)-Boligo, 0.3 µL of 1.25 μM R(G/A)-Boligo, 3 µL of fragment (the concentration of fragment is recommended in section “Barcoding”), and 4.6 µL of Blunt/TA Ligase Master Mix. The RE-digestion mixture was incubated at 37 °C for 1 h, and two microliters of the solution was used as template in PCR to amplify barcoded fragment by using corresponding PS-modified Aoligos. PCR product was purified and treated by using iodine solution as specified in section “PCR” and “Iodine-based chemical reaction”. All the non-modified oligos used to amplified the fragments with the 1-nt SEs are listed in Supplementary Data 1.

Publication protocol 1 Relevant paper

BfuI (BciVI) (5 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	VL PCR products (7.0 µL per reaction) were digested with 5 units of the restriction enzyme BciVI (BfuI) (Thermo Fisher Cat# ER1501) in a 20 µL reaction at 37°C for 2 hr. The enzyme was inactivated by heating to 80°C for 20 min.

Protocol tips
VL PCR products (7.0 µL per reaction) were digested with 5 units of the restriction enzyme BciVI (BfuI) (Thermo Fisher Cat# ER1501) in a 20 µL reaction at 37°C for 2 hr. The enzyme was inactivated by heating to 80°C for 20 min.

Manufacturer protocol Publication protocol 1 Relevant paper

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