<noautolink> ---+ Pulsed Field Gel Electrophoresis Mapping Purpose: To validate mutations predicted from whole-genome re-sequencing and possibly discover rearrangements through repetitive sequences or tandem duplications that are difficult to predict from sequencing data. ---++ Preparation of Plugs 1 Seal one side of block molds with tape. 1 Resuspend E. coli at 5 x 10<sup>8</sup> cells per ml. This will yield about 100 ng of DNA per well. 1 Prepare a 50-100 ml solution of 1% PFGE certified agarose dissolved in saline. This agarose stock can be melted and reused multiple times. Melt agarose and allow to cool to 50°C. 1 Add 500 µl of melted agarose stock cooled to 50°C to 500 µl of cell suspension in an eppendorf tube. Aliquot out ~800 µl into each space in the mold. 1 Allow gel plugs to solidify at 4°C for at least an hour. 1 Push gel plugs out of the mold into 50 ml conical tubes containing 16 ml of lysis buffer. Incubate overnight at 50°C in the hybridization oven. 1 Use a 10 ml pipet to remove lysis buffer. Add 16 ml fresh lysis buffer and incubate overnight at 37°C. 1 Add 16 ml storage buffer and incubate overnight at 37°C. ---++ Digestion with Restriction Enzyme Enzymes to use: =SfiI=, =NotI=, and =XbaI= cut an optimal number of times. =I-CeuI= and =AvrII= cut fewer times. 200 µl of volume needed per sample. ---+++ SfiI | *volume* | *component* | | 20 µl | NEB Buffer #2 | | 2 µl | 10x (1 mg/ml) BSA | | 176.5 µl | ddH<sub>2</sub>O | | 1.5 µl | SfiI, 20,000 U/ml (NEB) | | *200 µl* | *total* | Incubate overnight (16-24 hrs) at 50°C. ---+++ XbaI | *volume* | *component* | | 20 µl | NEB Buffer #2 | | 2 µl | 10x (1 mg/ml) BSA | | 176.5 µl | ddH<sub>2</sub>O | | 1.5 µl | XbaI, 20,000 U/ml (NEB) | | *200 µl* | *total* | Incubate overnight (16-24 hrs) at 37°C. ---+++ NotI | *volume* | *component* | | 20 µl | NEB Buffer #2 | | 2 µl | 10x (1 mg/ml) BSA | | 176.5 µl | ddH<sub>2</sub>O | | 1.5 µl | NotI, 10,000 U/ml (NEB) | | *200 µl* | *total* | Incubate overnight (16-24 hrs) at 37°C. ---++ Running Pulsed Field Gel ---++ Analysis of Gel
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Topic revision: r3 - 2009-08-17 - JeffreyBarrick