FastDigest Lsp1109I

Protocol tips

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	PCR products were column-purified using a nucleospin extract II kit (Macherey-Nagel) and recovered in 40 μl H20. Initial biotinylated di- and tri-blocks were digested with SfaNI (FastDigest, Fermentas); terminal di-modules were digested with BbvI (FastDigest Lsp1109I, Fermentas). Digested fragments were column purified and quantified (Nanodrop, Thermo scientific). Two micrograms of purified digested PCR products were typically obtained with this process.

Protocol tips

PCR products were column-purified using a nucleospin extract II kit (Macherey-Nagel) and recovered in 40 μl H20. Initial biotinylated di- and tri-blocks were digested with SfaNI (FastDigest, Fermentas); terminal di-modules were digested with BbvI (FastDigest Lsp1109I, Fermentas). Digested fragments were column purified and quantified (Nanodrop, Thermo scientific). Two micrograms of purified digested PCR products were typically obtained with this process.

Publication protocol

"The first step involves the construction of di- and tri-repeat block collections. Each mono repeat unit (HD:C, NG:T, NI:A, NN:G and NG*:T) was commercially synthesized on an individual basis (Top Gene Technologies) and subcloned in the pAPG10 plasmid (Top Gene Technologies). Each of the inserted sequences was flanked by BbvI and SfaNI type IIs restriction sites. The overhang created by these two enzymes was compatible except for the NG* (last half repeat unit) where SfaNI created a unique non-compatible overhang. In addition, a SfiI site was placed on the outside to allow recloning of every construction in the pAPG10 plasmid. All possible 16 di-, 64 tri-repeat units (excluding the one containing NG*) and 4 di-repeat units including NG* were prepared by consecutive restrictions, ligations (using BbvI and SfaNI) and subcloning in the pAPG10 using SfiI, creating a collection of 84 plasmids, which were PCR amplified to generate di/tri-modules for synthesis assembly.

For reverse synthesis, biotinylated di- and tri-blocks were amplified by PCR using the primers TAL-shuttle-F-short-Biotin (5′biotin-CCTCACAGGCCGGACGGGCCGAC-3′)/TAL-shuttle-R-short (5′-CCCGGTACCGCATCTCGAGG-3′). Terminal di-blocks (containing NG*) were amplified by PCR using the primers TAL-shuttle-F-short (5′-CCTCACAGGCCGGACGGGCCGAC-3′)/TAL-shuttle-R-short (5′-CCCGGTACCGCATCTCGAGG-3′). Conditions for PCR amplification were typically 5 ng of plasmid template, 250 μM dNTP mix, 200 nM of each oligonucleotide and 1 μl of Herculase II Fusion DNA Polymerase (Agilent) in a final volume of 50 μl of Herculase buffer 1 × . PCR conditions were 5 min at 95 °C, 30 cycles of 30 s at 95 °C, 30 s at 48 °C and 20 s at 72 °C and a final step of 3 min at 72 °C. PCR products were column-purified using a nucleospin extract II kit (Macherey-Nagel) and recovered in 40 μl H20. Initial biotinylated di- and tri-blocks were digested with SfaNI (FastDigest, Fermentas); terminal di-modules were digested with BbvI (FastDigest Lsp1109I, Fermentas). Digested fragments were column purified and quantified (Nanodrop, Thermo scientific). Two micrograms of purified digested PCR products were typically obtained with this process.

The assembly procedure started with two washing steps for the streptavidin-coated magnetic beads (5 μl, Dynabeads MyOne Streptavidin T1, Life Technologies) with 100 μl buffer A (TBS 1X, 0.05% TWEEN20) and was further performed as follows (all steps were performed in 50 μl under shaking, 700 r.p.m.): (1) parallel individual immobilization of the desired SfaNI-digested tri-blocks (100 ng) and di-blocks (70 ng) in buffer A on the streptavidin-coated magnetic beads, 1 h at room temperature followed by three washing steps with buffer B (TBS 1X, 0.05% TWEEN20, NaCl 1 M) and 2 with buffer A; (2) ligation of BbvI-digested terminal di-block (100 ng) in 1X ligation buffer containing 6U of T4 DNA ligase to the desired immobilized di- or tri-blocks at room temperature. After 30 min, 0.5 mM ATP (Fermentas, final concentration) was added and the reaction was set for an additional 30 min, followed by three washing steps with buffer B and 2 with buffer A; (3) release of the nascent chain from the solid surface by addition of BbvI (0.25 FDU, FastDigest Lsp1109I, Fermentas) in 1X Fast digestion buffer, followed by thermal inactivation (65 °C, 25 min) of the restriction enzyme. The reaction was subsequently cooled down to room temperature; (4) ligation of the nascent chain to the desired immobilized (step 1) di- or tri-blocks by addition of the reaction mixture from step 3 complemented with 1X ligation buffer and 6U of T4 DNA ligase. After 30 min, ATP was added as described in step 2; (5) steps 3 and 4 were repeated sequentially using the desired immobilized di- or tri- blocks; (6) after three washing steps with buffer B and 3 more with buffer A, the synthesized chain was released with either SfiI (New England Biolabs) to allow subcloning in the shuttle plasmid pAPG10, or by sequential SfaNI, wash, BbvI digestions to allow subcloning in plasmids already containing a TALEN scaffold. The product was then transformed in XL1b (Stratagene) according to standard molecular biology procedures; (7) selection of clones containing an insert of the desired size was achieved by colony PCR screening using appropriate primers, M13_Forward (5′-GTAAAACGACGGCCAG-3′)/M13_Reverse (5′-CAGGAAACAGCTATGAC-3′) or TAL-Screen-NForA (5′-GCGGGAGAGTTGAGAGGTCCAC-3′) / TAL-Screen-FRevA (5′-CAGGATACGGTCCTGGGTGCTGTTC-3′) for pAPG10 or TALEN backbone plasmid, respectively."

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Manufacturer protocol

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