FastDigest NotI

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	DNAs were then isolated using a NucleoSpin® 96 Flash (Macherey-Nagel) BAC DNA purification kit, digested with 5 U of FastDigest™ NotI enzyme (Fermentas) and size-fractioned by PFGE (6 V.cm−1, 5 to 15 s switch time, 16 h run time, 12.5 °C) in a Chef Mapper XA Chiller System 220 V (BioRad), followed by ethidium bromide staining and visualization.

Protocol tips
DNAs were then isolated using a NucleoSpin® 96 Flash (Macherey-Nagel) BAC DNA purification kit, digested with 5 U of FastDigest™ NotI enzyme (Fermentas) and size-fractioned by PFGE (6 V.cm−1, 5 to 15 s switch time, 16 h run time, 12.5 °C) in a Chef Mapper XA Chiller System 220 V (BioRad), followed by ethidium bromide staining and visualization.

Publication protocol

"BAC Selection and DNA Preparation
BAC clones were selected from the findings of Santos et al.15, which provides an initial overview of the P. edulis genome using BAC-end sequence (BES) data as a major resource. The results of comparative mapping between P. edulis’ BES and the reference genomes of Arabidopsis thaliana, Populus trichocarpa and Vitis vinifera were also used to choose BAC clones for sequencing. In addition, based on BES functional annotation results, the BAC-inserts with coding sequences (CDS) in one or both BESs were also selected.

A second selection procedure was performed after screening the genomic library using the probes homologous to P. edulis transcripts described in13. Briefly, the authors used suppression subtractive hybridization to construct two cDNA libraries enriched for transcripts induced and repressed by Xanthomonas axonopodis, respectively, 24 h after inoculation with a highly virulent bacterial strain.

The homologous probes were prepared via PCR, using as a template the genomic DNA from ‘IAPAR-123’, the accession used to construct the Ped-B-Flav BAC library. Specific primers were used to generate a single amplicon (200 to 600 bp in size) for each probe gene sequence. The ‘DecaLabel DNA Labeling Kit’ (Fermentas) was used for radiolabeling the probes. The amplification products were then purified with ‘Illustra ProbeQuantTM G-50 Micro Columns’ (GE Healthcare). The library was previously gridded onto macroarrays in which 41,472 clones were double-spotted on each 22 × 22 cm nylon membrane. These membranes were submerged in a bath of SSC (Saline-Sodium Citrate) solution (6×, 17 min., 50 °C); incubated overnight (68 °C) in hybridization buffer [6× SSC, 5× Denhardt’s Solution, 0.5% (w/v) SDS (Sodium Dodecyl Sulfate)]; hybridized with denatured probes (10 min, 95 °C; 1 min., cooled on ice); and washed twice in buffer 1 [2× SSC, 0.1% (w/v) SDS] (15 min., 50 °C) and buffer 2 [0.5× SSC, 0.1% (w/v) SDS] (30 min., 50 °C). Next, the hybridized membranes were placed in a film cassette for 24 h.; radioactive signals were detected using a PhosphorImagerTM and Storm 820 scanner (Amersham Biosciences) and analyzed using HDFR3 software, to identify the positive clones. Each positive clone was individually validated by PCR.

In order to estimate insert sizes, the preserved cultures were scraped and a positive single colony of each BAC grown in a 96-well plate (overnight, 37 °C) containing 1200 µL of LB medium with chloramphenicol (12.5 µg/mL) and glycerol (6%). DNAs were then isolated using a NucleoSpin® 96 Flash (Macherey-Nagel) BAC DNA purification kit, digested with 5 U of FastDigest™ NotI enzyme (Fermentas) and size-fractioned by PFGE (6 V.cm−1, 5 to 15 s switch time, 16 h run time, 12.5 °C) in a Chef Mapper XA Chiller System 220 V (BioRad), followed by ethidium bromide staining and visualization. The insert sizes were determined by comparison with PFGE (pulsed-field gel electrophoresis) standard size markers.

To prepare the DNA for sequencing, 1 μl of the above cultures was allowed to regrow in 20 mL of LB medium (plus 12.5 µg/mL chloramphenicol at 37 °C overnight) under shaking (250 rpm). The cultures were then mixed in pools, at a maximum of 20 clones per pool. DNA extraction was performed using the Nucleobond Xtra Midi Plus kit (Macherey-Nagel) according to the manufacturer’s instructions."

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