YAC library - Pulse Field Gel Electrophoresis

Thursday, December 04, 2003

Description
PFGE is needed for the separation of large DNA molecules, e.g. for separation of YACs from the yeast chromosomes. Using low melting point agarose the YAC can be isolated from the gel with the Gene Clean kit.
Markers: yeast chromosomes - AB1380 (used for library) or kit.

Procedure
PFGE is needed for the separation of large DNA molecules, e.g. for separation of YACs from the yeast chromosomes. Using low melting point agarose the YAC can be isolated from the gel with the Gene Clean kit.
Markers: yeast chromosomes - AB1380 (used for library) or kit.

Electrophoresis conditions:
CHEF pulse field gel apparatus
45 sec pulse times
125 V
Run time appr. 72 hrs.

Analytical Gel
agarose 1%
in 0.5 X TBE buffer

Southern Blot and Hybridization
Checking YAC's chromosome size and number with probe used for screening and isolation.

Preparative Gel
If YAC co-migrates with yeast chromosome on analytical PFGE: pulse time and/or volt can be changed 45 sec- 60 sec, 125v-145v respectively for runing the preparative gel.
agarose 1%
in 0.5 X TBE buffer

after solidify cut "window" and pour:
low melting agarose 1%
in 0.5X TBE buffer

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YAC library - Pulse Field Gel Electrophoresis