Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 7)

1. Inoculate a single colony into 50 to 100 ml YPD broth or
appropriate media. Grow with agitation to an O.D.
600
of
>1.0 at the appropriate temperature for your strain.
See Appendix for YPD broth.
2. When the desired O.D.
600
is reached, centrifuge the cells
at 5,000 x g, 10 min, 4 °C. Pour off the supernatant and
resuspend in 10 ml cold 50 mM EDTA, pH 8.
3. Determine the cell concentration by adding 10 µl of cells
to 990 µ l of water. Place the yeast suspension on a
hemocytometer and count at 400x power. See Appendix
on hemocytometer usage.
4. Heat the 2% CleanCut agarose solution using a
microwave and equilibrate the solution to 50 °C in a
water bath.
5. Calculate the amount of Cell Suspension Buffer and
CleanCut agarose necessary (see Section 9.2). For a
final concentration of 0.75% agarose use 0.63 ml of
Cell Suspension Buffer per ml of agarose plugs (use
100 µ l/plug for disposable mold or 300 µ l/plug for
reusable mold). Use 0.37 ml of 2% CleanCut agarose
per ml of agarose plugs to be made.
6. Remove 6 x 10 8 cells for each ml of plugs to be made.
Centrifuge in a microfuge for 3 minutes if volumes are
small, otherwise centrifuge the cells at 5,000 x g, for
10 minutes at 4 °C. Resuspend the cells in the volume of
Cell Suspension Buffer calculated above and equilibrate
to 50 °C.
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10. Wash the plugs four times in 1x Wash Buffer, 1 hour each
at room temperature with gentle agitation (for each plug,
use 1 ml of 1x Wash Buffer). Prepare the Wash Buffer by
diluting the 10x stock (1:10) with sterile ddH
2
O. If the
plugs are to be used in subsequent enzyme reactions, it is
advisable to wash the plugs in 1 mM PMSF during the
second or third wash to inactivate residual Proteinase K.
See Appendix for PMSF stock solution.
Note: For washing a few plugs, use a 2 or 5 ml sterile tube.
11. Store the plugs at 4 °C in 1x Wash Buffer. The plugs
should be stable for 3 months.
Section 4
Preparation of Agarose Embedded
Yeast DNA
Reagents and Equipment Needed
50 mM EDTA, pH 8
Sterile transfer pipettes
50 °C water bath
Microscope
PMSF stock solution see Appendix
Hemocytometer see Appendix
2, 5, 50 ml sterile plastic tubes
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