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.
2. Resuspend E. coli at 5 x 10⁸ cells per ml. This will yield about 100 ng of DNA per well.
3. 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.
4. 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.
5. Allow gel plugs to solidify at 4°C for at least an hour.
6. 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.
7. Use a 10 ml pipet to remove lysis buffer. Add 16 ml fresh lysis buffer and incubate overnight at 37°C.
8. 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

Incubate overnight (16-24 hrs) at 50°C.

XbaI

Incubate overnight (16-24 hrs) at 37°C.

NotI

Incubate overnight (16-24 hrs) at 37°C.

Running Pulsed Field Gel

Analysis of Gel