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 50C.
  4. Add 500 l of melted agarose stock cooled to 50C to 500 l of cell suspension in an eppendorf tube. Aliquot out ~800 l into each space in the mold.
  5. Allow gel plugs to solidify at 4C 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 50C in the hybridization oven.
  7. Use a 10 ml pipet to remove lysis buffer. Add 16 ml fresh lysis buffer and incubate overnight at 37C.
  8. Add 16 ml storage buffer and incubate overnight at 37C.

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 l of volume needed per sample.

SfiI

volume component
20 l NEB Buffer #2
2 l 10x (1 mg/ml) BSA
176.5 l ddH2O
1.5 l SfiI, 20,000 U/ml (NEB)
200 l total

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

XbaI

volume component
20 l NEB Buffer #2
2 l 10x (1 mg/ml) BSA
176.5 l ddH2O
1.5 l XbaI, 20,000 U/ml (NEB)
200 l total

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

NotI

volume component
20 l NEB Buffer #2
2 l 10x (1 mg/ml) BSA
176.5 l ddH2O
1.5 l NotI, 10,000 U/ml (NEB)
200 l total

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

Running Pulsed Field Gel

Analysis of Gel

 Barrick Lab  >  ProtocolList  >  ProceduresPulsedFieldGelElectrophoresisMapping

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