MboII NEB#R0148S

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	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.

Protocol tips

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.

Publication protocol

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.

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