Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 4)

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
7 cells for each ml of agarose
plugs to be made (use 100 µl/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.
3
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 µl 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
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,
50 well disposable plug mold, 2; Screened Cap, 1
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, 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
2
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