Bio-Rad CHEF Genomic DNA Plug Kits User Manual
CHEF Genomic DNA Plug Kits
Instruction Manual
Catalog Numbers
170-3591
170-3592
170-3593
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Table of Contents
Section 1 Introduction......................................................1
1.1 Kit Components ..........................................................2
Section 2 Preparation of Agarose Embedded
Section 3 Preparation of Agarose Embedded
Section 4 Preparation of Agarose
Section 5 Restriction Enzyme Digestion of Plugs........11
Section 6 References ......................................................12
Section 7 Additional Reagents for Pulsed Field
Section 8 Instruments for Pulsed Field
Section 9 Appendix ........................................................14
Mammalian DNA ............................................3
Bacterial DNA..................................................5
Embedded Yeast DNA ....................................8
Electrophoresis ..............................................13
Electrophoresis ..............................................13
9.1 Solutions....................................................................14
9.2 Agarose Concentrations ............................................15
9.3 Hemocytometer Usage ..............................................16
Section 1
Introduction
Pulsed-Field Gel Electrophoresis (PFGE) allows the
separation of DNA ranging in size from a few kilobase pairs
to 10 megabase pairs. Because of the large size of these
molecules, simple pipetting mechanically shears the DNA
resulting in unacceptable quality for PFGE separations. This
has necessitated procedures for lysis of whole cells embedded
in agarose, allowing purification of chromosome-sized DNA
without shearing.
The most important and difficult task in preparing cells
for embedding in agarose is to obtain the proper cell
concentration. Although optical density is frequently used to
determine cell concentration, it is not reliable. Different
strains, plasmid content, and growth media all contribute to
the actual cell number achieved for a particular optical
density. Variation in cell number will cause the amount of
DNA per agarose plug to vary, leading to over- and or underloading of the sample. To eliminate the need to generate a
growth curve for each strain, a hemocytometer is the most
reproducible method for achieving the proper cell
concentration for different types of cells, bacteria, yeast, and
fungi. Detailed instructions for the use of a hemocytometer
are given in the Appendix.
1
1.1 Kit Components
The CHEF Genomic DNA Plug Kits are designed to
produce 100 sample plugs of agarose embedded DNA with
the quality necessary for PFGE separations. All kits consist
of a core module, which contains all the buffers and
proteinase solutions. This core module is the only component
supplied with the mammalian DNA kit. Other kits contain
additional modules with lysis buffers specific to that cell
type. For example, the bacterial kit contains lysozyme and its
reaction buffer necessary to digest the outer cell membrane of
lysozyme-sensitive bacteria; the yeast module contains
lyticase and its reaction buffer necessary to digest the cell
walls of most yeast. Each of the disposable plug molds
provided contains 50 wells which are 1.5 mm thick and 5 mm
wide and hold 85 µ l of volume. It is also possible to use the
10 well reusable sample plug mold (170-3622). Each of the
wells in the reusable mold are 1 cm wide and 1.5 cm thick
and hold 300 µl of volume.
Catalog
Number Product Description
170-3591 CHEF Mammalian Genomic DNA Plug Kit, contains
170-3592 CHEF Bacterial Genomic DNA Plug Kit, contains
Cell Suspension Buffer, 12 ml; >600 U/ml Proteinase K,
1.3 ml; Proteinase K Reaction Buffer, 30ml;
2% CleanCut
50 well disposable plug mold, 2; Screened Cap, 1
Cell Suspension Buffer, 12 ml; >600 U/ml Proteinase K,
1.3 ml; Proteinase K Reaction Buffer, 30 ml;
2% CleanCut Agarose, 12 ml; 10x Wash Buffer, 60 ml;
25 mg/ml Lysozyme, 1.6 ml; Lysozyme Buffer, 30 ml,
50 well disposable plug mold, 2; Screened Cap, 1
™
Agarose, 12 ml; 10x Wash Buffer, 60 ml,
170-3593 CHEF Yeast Genomic DNA Plug Kit, contains Cell
Suspension Buffer, 12 ml; >600 U/ml Proteinase K,
1.3 ml; Proteinase K Reaction Buffer, 30 ml;
2% CleanCut Agarose, 12 ml; 10x Wash Buffer, 60 ml;
5000 U/ml Lyticase, 1.6 ml; Lyticase Buffer, 30 ml,
50 well disposable plug mold, 2; Screened Cap, 1
Section 2
Preparation of Agarose Embedded
Mammalian DNA
Reagents and Equipment Needed
Sterile transfer pipettes
50 °C water bath
PMSF stock solution see Appendix
Hemocytometer see Appendix
Microscope
2, 5, 50 ml sterile plastic tubes
1. Prepare a cell suspension in isotonic saline or tissue
culture medium without fetal bovine serum. Count the
cells and remove 5 x 10
plugs to be made (use 100 µ l/plug for disposable mold or
300 µ l/plug for reusable mold) and place on ice.
See Appendix for hemocytometer usage.
2. Melt the 2% CleanCut agarose solution using a
microwave and equilibrate the solution to 50 °C in a
water bath.
7
cells for each ml of agarose
2
3
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