Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 9)

Section 7
Additional Reagents for
Pulsed Field Electrophoresis
Catalog
Number
Product Description
162-0137 Pulsed Field Certified Agarose, 100 g
162-0135 Chromosomal Grade Agarose, 25 g
170-3594 CleanCut Agarose, 2%, 12 ml
170-3624 DNA Size Standards, 5 kb ladder
170-3605 DNA Size Standards, Yeast chromosomal
170-3633 DNA Size Standards, S. pombe chromosomes
170-3635 DNA Size Standards, lambda ladder
162-0196 Zeta-Probe ® GT Nylon Membrane, roll, 30 cm x 3.3 m
161-0733 10x TBE Buffers, 1 liter
Section 8
Instruments for Pulsed Field
Electrophoresis
Catalog
Number
Product Description
170-3612 CHEF-DR ® II System
170-3695 CHEF-DR III System
170-3670 CHEF Mapper ® XA Chiller System
170-3654 Cooling Module
165-5031 GS GeneLinker ® UV Chamber
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2. Aspirate the Wash Buffer and add 1 ml of the appropriate
1x restriction enzyme buffer for about 1 hour with gentle
agitation at room temperature. Aspirate off the buffer and
add 0.3 ml of fresh 1x enzyme buffer. Add the restriction
enzyme (30–50 U per 100 µl plug) and incubate overnight
at the appropriate temperature.
3. After overnight digestion, remove the buffer and incubate
in 1 ml of 1x Wash Buffer for approximately 30 minutes
with gentle agitation.
4. Optional: Remove the Wash Buffer and equilibrate the
plug in the appropriate concentration of gel running
buffer, i.e. 0.5x TBE, 1.0x TAE, etc.
5. Load 1 ⁄ 4 to 1 ⁄ 3 of a plug per well (this is approximately 50 µg
DNA) and adjust the volume if necessary on subsequent
gels. In addition, always load appropriate size standards.
Section 6
References
1. Smith, C. L., Klco, S. R. and Cantor, C. R., Genome Analysis,
Chapter 3, K. Davis ed., IRL Press Ltd., Oxford, England (1988).
2. Smith, C. L. and Cantor, C. R., Methods in Enzymology, 155,
449–467 (1988).
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