Barrick Lab :: ProceduresPulsedFieldGelElectrophoresisMapping

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---+ 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

Barrick Lab > ProtocolList > ProceduresPulsedFieldGelElectrophoresisMapping

Contributors to this topic

JeffreyBarrick

Topic revision: r3 - 2009-08-17 - 22:40:14 - Main.JeffreyBarrick