104
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
Components of a
pulsed-field gel
experiment
structing long-range restriction maps of chromosomes, PFGE has been also
used as a preparative tool (Michiels et al. 1987).
Additional information on PFGE procedures can be found in books on
PFGE methodology (e.g., Burmeisterand Ulanovsky 1992; Birren and Lai
1993; Anand 1992).
Materials
• DNA agarose blocks made from specific cells, digested with infrequently
cleaving restriction enzymes
• DNA size markers: Yeast chromosomes and lambda concatemers
• Agarose gel
• PFGE buffer
• OF AGE, FIGE, CHEF or RüFE apparatus with switching unit and cooling
supply
Subprotocol 1
Preparation and Processing of High Molecular DNA in Agarose Blocks
To protect DNA from shearing, high molecular weight DNA is prepared
from cells embedded in agarose blocks. In principle, DNA can be derived
from single cell suspensions of all different tissues or celllines; most commonly blood is used.
Materials
Reagents • Whole blood, solid organ, cell line
• Agarose (LMP and normal agarose)
• PMSF (phenylmethylsulfonylfluoride)
• Proteinase K
• EDTA
• Sodium lauroyl sarcosinate
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, ERCOLE RAO AND BIRGIT WEISS
Components of a
pulsed-field gel
experiment
structing long-range restriction maps of chromosomes, PFGE has been also
used as a preparative tool (Michiels et al. 1987).
Additional information on PFGE procedures can be found in books on
PFGE methodology (e.g., Burmeisterand Ulanovsky 1992; Birren and Lai
1993; Anand 1992).
Materials
• DNA agarose blocks made from specific cells, digested with infrequently
cleaving restriction enzymes
• DNA size markers: Yeast chromosomes and lambda concatemers
• Agarose gel
• PFGE buffer
• OF AGE, FIGE, CHEF or RüFE apparatus with switching unit and cooling
supply
Subprotocol 1
Preparation and Processing of High Molecular DNA in Agarose Blocks
To protect DNA from shearing, high molecular weight DNA is prepared
from cells embedded in agarose blocks. In principle, DNA can be derived
from single cell suspensions of all different tissues or celllines; most commonly blood is used.
Materials
Reagents • Whole blood, solid organ, cell line
• Agarose (LMP and normal agarose)
• PMSF (phenylmethylsulfonylfluoride)
• Proteinase K
• EDTA
• Sodium lauroyl sarcosinate