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Results: 7

1.
<b>Figure 3</b>

Figure 3. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

An illustration of the conditionally toxic selection vector ptoxBAC832 prepared by the MapDraw program of DNASTAR Lasergene. GCT denotes the sequence 5′-GCTGCCGC-3′. GCA denotes the sequence 5′-GCAGCCGC-3′. GCC denotes the sequence 5′-GCCGCCGC-3′. GCGT denotes the sequence 5′-GCGTCCGC-3′.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.
2.
<b>Figure 6</b>

Figure 6. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

Competitive cleavage of two linear substrates by variant E156K/M91V. The 2686 bp substrate was derived from pUC-GCT and the 1778 bp substrate was derived from pUC-NotI. Equimolar amounts of each substrate were mixed and incubated with increasing concentrations of enzyme. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer. (−) indicates no enzyme addition.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.
3.
<b>Figure 5</b>

Figure 5. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

Variant M91V/E156K digestion of plasmid substrate pUC-GCT. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1 × NEB BamHI buffer. (−) indicates no enzyme addition.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.
4.
<b>Figure 2</b>

Figure 2. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

Variant E156K digestion of a substrate containing a single 5′-GCTGCCGC-3′ site. Plasmid pUC-GCT was derived from pUC19. The enzyme/substrate (E/S) molar ratio is given above each lane. Lane M, 1 kb DNA ladder. All reactions were incubated at 37°C for 60 min in 1× NEB BamHI buffer.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.
5.
<b>Figure 1</b>

Figure 1. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

Altered cleavage characteristics of NotI variant E156K determined by digestion of plasmid substrate pXba. Lane 1, no enzyme addition (−); Lane 2, digestion with 100 U wt NotI; Lanes 3–9, increasing amounts of variant E156K. Lane M, 1 kb DNA ladder (NEB). All reactions were incubated at 37°C for 60 min in 1× NEB buffer 3.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.
6.
<b>Figure 7</b>

Figure 7. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

Digestion of T7 genomic DNA by variant M91V/E156K. (A) A virtual digest of T7 DNA using NEBcutter (29). Since site 5′-GCGTCCGC-3′ is nicked by this enzyme it was not included in the virtual digest. M is a size marker lane. (B) Actual results of digesting T7 DNA in 1× NEB BamHI buffer for various times at 37°C. A 30-fold molar excess of enzyme was used in each reaction. (C) A control showing no digestion of T7 DNA by 200 U wt NotI.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.
7.
<b>Figure 4</b>

Figure 4. From: Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.

Evaluation of double variants by digestion of pBR322 (linearized by AflIII). Lane M, 1 kb DNA ladder; Lane 1, incubation with 100 U wt NotI to confirm no cleavage of pBR322; Lane 2, incubation with cell extract from clone EP1 (E156K/V348M); Lane 3, incubation with cell extract from clone EP5 (E156K/F157C); Lane 4, incubation with cell extract from clone EP10 (M91V/E156K); Lane 5, incubation with cell extract containing variant E156K. All reactions were at 37°C for 60 min in 1× NEB buffer 3. Production of 2.9 and 1.5 kb fragments is consistent with cleavage at 5′-TCGGCCGC-3′.

James C. Samuelson, et al. Nucleic Acids Res. 2006;34(3):796-805.

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